Localized use of nitric oxide-adducts to prevent internal tissue damage

ABSTRACT

A method for preventing adverse effects associated with the use of a medical device in a patient by introducing into the patient a device of which at least a portion includes a prophylactic or therapeutic amount of a nitric oxide adduct. The nitric oxide adduct can be present in a matrix coating on a surface of the medical device; can be coated per se on a surface of the medical device; can be directly or indirectly bound to reactive sites on a surface of the medical device; or at least a portion of the medical device can be formed of a material, such as a polymer, which includes the nitric oxide adduct. Also disclosed is a method for preventing adverse effects associated with the use of a medical device in a patient by introducing the device during a medical procedure and before or during said procedure locally administering a nitric oxide adduct to the site of contact of said device with any internal tissue.

This application is a continuation-in-part of copending U.S. applicationSer. No. 08/123,331, filed Sep. 17, 1993, now abandoned.

This invention relates to the use of medical devices and to thetreatment of damaged vasculature. More particularly, the inventionrelates to the use of medical devices which are inserted into a patientwherein at least a portion of the device includes a surface whichexposes and delivers a form of nitric oxide to vascular surfaces withwhich it comes in contact. Alternatively the invention relates to thefield of preventing the adverse effects which result from medicalprocedures which involve the use of such a medical device and whichinclude administering a source of nitric oxide to the cite ofvasculature contact of such medical devices.

The vascular endothelium participates in many homeostatic mechanismsimportant for the regulation of vascular tone and the prevention ofthrombosis. A primary mediator of these functions is endothelium-derivedrelaxing factor (EDRF). First described in 1980 by Furchgott andZawadzki (Furchgott and Zawadzki, Nature (Lond.). 288:373-376, 1980)EDRF is either nitric oxide (Moncada et al., Pharmacol Rev. 43:109-142,1991.) (NO) or a closely related NO-containing molecule (Myers et al.,Nature (Lond.), 345:161-163, 1990).

Removal of the endothelium is a potent stimulus for neointimalproliferation, a common mechanism underlying the restenosis ofatherosclerotic vessels after balloon angioplasty. (Liu et al.,Circulation, 79:1374-1387, 1989); (Fems et al., Science, 253:1129-1132,1991). Nitric oxide dilates blood vessels (Vallance et al., Lancet,2:997-1000, 1989) inhibits platelet activation and adhesion (Radomski etal., Br. J Pharmacol, 92:181-187, 1987) and, in vitro, nitric oxidelimits the proliferation of vascular smooth muscle cells (Garg et al.,J. Clin. Invest., 83:1774-1777, 1986). Similarly, in animal models,suppression of platelet-derived mitogens decreases intimal proliferation(Fems et al., Science, 253:1129-1132, 1991). The potential importance ofendothelium-derived nitric oxide in the control of arterial remodelingafter injury is further supported by recent preliminary reports inhumans suggesting that systemic NO donors reduce angiographic restenosissix months after balloon angioplasty (The ACCORD Study Investigators, J.Am. Coll. Cardiol. 23:59A. (Abstr.), 1994).

Biologic thiols react readily with NO (probably as N₂ O₃ or NO) underphysiologic conditions to form stable, biologically activeS-nitrosothiol species (Stamler et al., Proc. Natl. Acad. Sci. U S A.,89:444-448, 1992). S-nitrosothiols exhibit EDRF-like activity in vitroand in vivo, including vasodilation (Myers et al., Nature (Lond.),345:161-163, 1990) and platelet inhibition via a cyclic 3',5'-guanosinemonophosphate (cGMP)-dependent Mechanism (Loscalzo, J. Clin. Invest.,76:703-708, 1985); (Keaney et al., J. Clin. Invest., 91:1582-1589,1993).

Over the past two decades, much research effort has been directedtowards the development of medical devices and machines that are used ina wide variety of clinical settings to maintain the vital physiologicalfunctions of a patient. For example, such devices as catheters,prosthetic heart valves, arteriovenous shunts and stents are usedextensively in the treatment of cardiac and other diseases.

However, platelet deposition on artificial surfaces severely limits theclinical usefulness of such devices. Forbes et al., Brit. Med. Bull.34(2):201-207, 1978; Sheppeck et al., Blood, 78(3):673-680, 1991. Forexample, exposure of blood to artificial surfaces frequently leads toserious thromboembolic complications in patients with artificial heartvalves, synthetic grafts and other prosthetic devices, and in patientsundergoing external circulation, including cardiopulmonary bypass andhemodialysis. Salzman, Phil. Trans. R. Soc. Lond., B294:389-398, 1981.

The normal endothelium which lines blood vessels is uniquely andcompletely compatible with blood. Endothelial cells initiate metabolicprocesses, like the secretion of prostacyclin and endothelium-derivedrelaxing factor (EDRF), which actively discourage platelet depositionand thrombus formation in vessel walls. No material has been developedthat matches the blood-compatible surface of the endothelium. In fact,in the presence of blood and plasma proteins, artificial surfaces are anideal setting for platelet deposition (Salzman et al., supra, 1981).Exposure of blood to an artificial surface initiates reactions that leadto clotting or platelet adhesion and aggregation. Within seconds ofblood contact, the artificial surface becomes coated with a layer ofplasma proteins which serves as a new surface to which platelets readilyadhere, become activated, and greatly accelerate thrombus formation(Forbes et al., supra, 1978).

This creates problems in the use of artificial materials at themicrovascular level, where the ratio of vessel surface area to bloodvolume is high (Sheppeck et al., supra). For example, thromboembolism isstill the most serious complication following prosthetic heart valveimplantation, despite changes in design and materials used. In fact, theincidence of detectable thromboembolism can be as high as 50%, dependingon the valve design and construction (Forbes et al.). Further,cardiopulmonary support systems used during cardiac surgery areresponsible for many of the undesirable hemostatic consequences of suchsurgery (Bick, Semin. Thromb. Hemost. 3:59-82, 1976). Thrombosis is alsoa significant problem in the use of prosthetic blood vessels,arteriovenous shunts, and intravenous or intraarterial catheters.

Conventional methods for preventing thrombus formation on artificialsurfaces have a limited effect on the interaction between blood andartificial surfaces. For example, in cardiopulmonary bypass andhemodialysis heparin has little effect, and the only platelet reactionsinhibited by anticoagulants are those induced by thrombin. In fact, itseems that heparin actually enhances the aggregation of platelets(Salzman et al., J. Clin. Invest., 65:64, 1980). To further complicatematters, heparin when given systemically, can accelerate hemorrhage,already a frequent complication of cardiac surgery.

Attempts to inhibit platelet deposit on artificial surfaces involvesystemic administration of aspirin, dipyridamole, and sulfinpyrazone.While these have some effect in preventing thromboembolism when givenwith oral anticoagulants, serious adverse effects can result. Blood lossis significantly increased in bypass or hemodialysis patients followingadministration of aspirin (Torosian et al., Ann. Intern. Med.89:325-328, 1978). In addition, the effect of aspirin and similarlyacting drugs is not promptly reversible, which is essential duringcardiopulmonary bypass. Finally, agents such as aspirin, which depressplatelet function by inhibiting cyclo-oxygenase, may block plateletaggregation, but they do not prevent the adhesion of platelets toartificial surfaces (Salzman et al., supra, 1981).

Despite considerable efforts to develop non-thrombogenic materials, nosynthetic material has been created that is free from this effect. Inaddition, the use of anticoagulant and platelet-inhibiting agents hasbeen less than satisfactory in preventing adverse consequences resultingfrom the interaction between blood and artificial surfaces.Consequently, a significant need exists for the development ofadditional methods for preventing platelet deposition and thrombusformation on artificial surfaces.

In the same manner as artificial surfaces, damaged arterial surfaceswithin the vascular system are also highly susceptible to thrombusformation. The normal, undamaged endothelium prevents thrombus formationby secreting a number of protective substances, such asendothelium-derived relaxing factor (EDRF), which prevents bloodclotting primarily by inhibiting the activity of platelets. Diseasestates such as atherosclerosis and hyperhomocysteinemia cause damage tothe endothelial lining, resulting in vascular obstruction and areduction in the substances necessary to inhibit blood clotting. Thus,abnormal platelet deposition resulting in thrombosis is much more likelyto occur in vessels in which endothelial damage has occurred. Whilesystemic agents have been used to prevent coagulation and inhibitplatelet function, a need exists for a means by which a damaged vesselcan be treated directly to prevent thrombus formation.

Balloon arterial injury results in endothelial denudation and subsequentregrowth of dysfunctional endothelium (Saville, Analyst, 83:670-672,1958) that may contribute to the local smooth muscle cell proliferationand extracellular matrix production that result in reocclusion of thearterial lumen.

Reported work on platelet aggregation has demonstrated the effect ofnitric oxide adducts on the inhibition of platelet-to-plateletaggregation as a specific stage in clot formation that relates to theircommon interaction with each other.

Toward arriving at the present invention, the inventors hypothesizedthat local delivery of an EDRF-like species to restore or replace thedeficiency in EDRF noted with dysfunctional endothelium will modulatethe effects of vascular injury and reduce intimal proliferationfollowing injury. The observations that form the basis of this inventionrelate to the active deposition of platelets on non-platelet tissue bedsrather than platelet-to-platelet aggregation.

In accordance with an aspect of the present invention, there is provideda process and product for preventing adverse effects associated with theuse of a medical device in a patient wherein at least a portion of thedevice includes a nitric oxide adduct. Such adverse effects include butare not limited to platelet adhesion and/or thrombus formation when themedical device is used in a blood vessel. As known in the art, plateletadhesion and subsequent platelet activation may result in the blockageof blood vessels particularly after procedures involving use of amedical device for removing blockages such as those often referred to asthe phenomenon of restenosis. The medical device can be used elsewhere,such as for example, in patients having cancer of the gastrointestinaltract in the Sphincter of Oddi where indwelling stents (e.g., aPalmaz-Schatz stent, J&J, New Brunswick, N.J.) are placed to maintainpatency of the lumen. They are also used in patients having cancer ofthe esophagus to support the airway opening.

The medical device or instrument of the invention can be, for example, acatheter, prosthetic heart valve, synthetic vessel graft, stent (e.g.,Palmaz-Schatz stent), arteriovenous shunt, artificial heart, intubationtubes, airways and the like.

As noted above, in this aspect the device is provided a nitric oxideadduct. Thus, for example, (i) all or a portion of the medical devicemay be coated with a nitric oxide adduct, either as the coating per seor in a coating matrix; (ii) all or a portion of the medical device maybe produced from a material which includes a nitric oxide adduct, forexample, a polymer which has admixed therewith a nitric oxide adduct orwhich includes as pendent groups or grafts one or more of such nitricoxide adducts; or (iii) all or a portion of the tissue-contractingsurfaces of the medical device may be derivatized with the nitric oxideadduct.

In the first embodiment of the above aspect, coatings can be ofsynthetic or natural matrices, e.g. fibrin or acetate-based polymers,mixtures of polymers or copolymers, respectively. Preferably they arebioresorbable or biodegradable matrices. Such matrices can also providefor metered or sustained release of the nitric oxide adduct. The devicesurfaces can be substituted with or the coating mixture can furtherinclude other medicaments, such as anticoagulants and the like.

In the next embodiment of this aspect, nitric oxide adducts areincorporated into the body of a device which is formed of abiodegradable or bioresorbable material. Thus, intact nitric oxideadduct is released over a sustained period of the resorption ordegradation of the body of the device.

In the embodiment relating to the derivatization of an artificialsurface, such as of a medical device or instrument with a nitric oxideadduct, the artificial surfaces may be composed of organic materials ora composite of organic and inorganic materials. Examples of suchmaterials include but are not limited to synthetic polymers orcopolymers containing nitric oxide adducts, gold or coated metalsurfaces upon which a functionalized monolayer containing the nitricoxide adduct is adsorbed, or synthetic polymeric materials or proteinswhich are blended with nitric oxide adducts.

Another principal aspect of the invention relates to a medical devicecomprising an instrument suitable for introduction into a patient ofwhich at least a portion comprises a nitric oxide adduct. As withrespect to the above method, (i) all or a portion of the medical devicemay be coated with a nitric oxide adduct, either as the coating per seor in a coating matrix; (ii) all or a portion of the medical device maybe produced from a material which includes a nitric oxide adduct, forexample, a polymer which has admixed therewith a nitric oxide adduct orwhich includes as pendent groups or grafts one or more of such nitricoxide adducts; or (iii) all or a portion of the tissue-contactingsurfaces of the medical device may be derivatized with the nitric oxideadduct.

Again, the medical device or instrument of the invention can be, forexample, a catheter, prosthetic heart valve, synthetic vessel graft,stent, arteriovenous shunt, artificial heart, intubation tube andairways and the like.

Another principal aspect of the invention relates to a method fortreating a damaged blood vessel surface or other injured tissue bylocally administering a nitric oxide adduct to the site of the damagedblood vessel. Such damage may result from the use of a medical device inan invasive procedure. Thus, for example, in treating vasculatureblocked, for example by angioplasty, damage can result to the bloodvessel. Such damage may be treated by use of a nitric oxide adduct. Inaddition to repair of the damaged tissue, such treatment can also beused to prevent and/or alleviate and/or delay reocclusions, for examplerestenosis. Preferably, all or most of the damaged area is coated withthe nitric oxide adduct per se or in a pharmaceutically acceptablecarrier or excipient which serves as a coating matrix. This coatingmatrix can be of a liquid, gel or semisolid consistency. The nitricoxide adduct can be applied in combination with other therapeuticagents, such as antithrombogenic agents. The carrier or matrix can bemade of or include agents which provide for metered or sustained releaseof the therapeutic agents. Nitric oxide adducts which are preferred foruse in this aspect are mono-or poly-nitrosylated proteins, particularlypolynitrosated albumin or polymers or aggregates thereof. The albumin ispreferably human or bovine, including humanized bovine serum albumin.

The localized, time-related, presence of nitric oxide adductsadministered in a physiologically effective form is efficacious indiminishing, deterring or preventing vascular damage after or as aresult of instrumental intervention, such as angioplasty,catheterization or the introduction of a stent (e.g., Palmaz-Schatzstent) or other indwelling medical device.

Local administration of a stable nitric oxide adduct inhibits neointimalproliferation and platelet deposition following vascular arterialballoon injury. This strategy for the local delivery of a long-lived NOadduct is useful for the treatment of vascular injury followingangioplasty.

Typical nitric oxide adducts include nitroglycerin, sodiumnitroprusside, S-nitroso-proteins, S-nitroso-thiols, long carbon-chainlipophilic S-nitrosothiols, S-nitrosodithiols, iron-nitrosyl compounds,thionitrates, thionitrites, sydnonimines, furoxans, organic nitrates,and nitrosated amino acids.

Particularly preferred is the localized use of nitroso-proteins,particularly those which do not elicit any significant immune response.An example of such a nitroso-protein which does not elicit anysignificant immune response is a mono- or poly-nitrosated albumin. Suchnitrosylated albumins, particularly the polynitrosylated albumins, canbe present as polymeric chains or three dimensional aggregates where thepolynitrosylated albumin is the monomeric unit. The albumin of themonomeric unit can be a functional subunit of full-length native albuminor can be an albumin to which has been attached an additional moiety,such as a polypeptide, which can aid, for example, in localization. Theaggregates are multiple inter adherent monomeric units which canoptionally be linked by disulfide bridges. Additionally devices whichhave been substituted or coated with nitroso-protein have the uniqueproperty that they can be dried and stored.

An additional particularly unique aspect of the invention is that thiscontemplates "recharging" the coating that is applied to a device, suchas a catheter or other tubing as considered above, by infusing a nitricoxide donor to a previously coated surface. For example, anS-nitroso-protein such as S-nitroso albumin will lose its potency invivo as the NO group is metabolized, leaving underivatized albumin.However, it has been recognized by the inventors that the surfacecoating can be "recharged" by infusing an NO donor such asnitroprusside. This principal is demonstrated by the experimentsreported in Example 2 in which nitroprusside is mixed with albuminengendering subsequent protection against platelet deposition.

Another aspect of the invention is related to the derivatization of anarticial surface with a nitric oxide adduct for preventing the depositof platelets and for preventing thrombus formation on the artificialsurface. The artificial surfaces may be composed of organic materials ora composite of organic and inorganic materials. Examples of suchmaterials include but are not limited to synthetic polymers orcopolymers containing nitric oxide adducts, gold or gold coated metalsurfaces upon which a functionalized monolayer containing the nitricoxide adduct is adsorbed, or synthetic polymeric materials or proteinswhich are blended with nitric oxide adducts.

The invention also relates to a method and product for administering anitric oxide adduct in combination with one or more anti-thrombogenicagents. Such agents include heparin, warfarin, hirudin and its analogs,aspirin, indomethacin, dipyridamole, prostacyclin, prostaglandin El,sulfinpyrazone, phenothiazines (such as chlorpromazine ortrifluperazine) RGD (arginine-glycine-aspartic acid) peptide or RGDpeptide mimetics, (See Nicholson et al., Thromb. Res., 62:567-578,1991), agents that block platelet glycoprotein IIb-IIIa receptors (suchas C-7E3), ticlopidine or the thienopyridine known as clopidogrel.

Other therapeutic agents can also be included in the coating or linkedto reactive sites in or on the body of the device. Examples of theseinclude monoclonal antibodies directed towards certain epitopes/ligandssuch as platelet glycoprotein IIb/IIIa receptor or cell adhesionmolecules such as the CD-18 complex of the integrins or PECAM-1;fragments of recombinant human proteins eg, albumin; pegylated proteins;anti-sense molecules; viral vectors designed as vehicles to delivercertain genes or nucleoside targeting drugs.

The invention will now be further described by reference to a briefdescription of each of the Figures, but in no way are a limitation ofthe scope of the invention.

FIG. 1A is a synthetic scheme for the preparation of a nitrosothiolincorporated on to the ε-amino group of a copolymer comprised ofpoly-L-lactic acid-co-lysine.

FIG. 1B is a synthetic scheme for the preparation of a nitrosothiolincorporated on to the ε-amino group of a copolymer comprised ofpoly-L-lactic acid-co-L-lysine.

FIG. 2A is a synthetic scheme for the preparation of a nitrosothiolincorporated on to a carboxylate derivatized self-assembled monolayer(SAMS) adsorbed to a gold surface.

FIG. 2B is a synthetic scheme for the preparation of a nitrosothiolincorporated on to an amino derivatized self-assembled monolayer (SAMS)adsorbed to a gold surface.

FIG. 3 is a plot demonstrating [¹²⁵ I]-labeled S-nitroso-albumin ([¹²⁵I]-S-NO-BSA) binding to injured rabbit femoral artery as a function ofthe method of delivery. Rabbit femoral arteries were isolated andballoon-injured as described in Example 1 and [¹²⁵ I]-S-NO-BSA appliedeither directly into the injured artery (local) or injectedintraarterially via the opposite femoral artery (systemic). [¹²⁵I]-S-NO-BSA binding was determined by quantification of radioactivityafter flow was reestablished for a period of 15 minutes. Non-specific[¹²⁵ I]-S-NO-BSA binding (sham) was determined from uninjured carotidartery harvested simultaneously with femoral arteries. Data arepresented as mean +/- SEM per gram of wet tissue weight, and are derivedfrom four animals. *P<0.0.029, local vs. systemic delivery and +/+P<0.05, systemic injured vs. sham.

FIG. 4 is a plot demonstrating the effect of polythiolatedS-nitroso-albumin (pS-NO-BSA) and polythiolated albumin (pS-BSA) on [¹¹¹In]-labeled platelet binding to injured rabbit femoral arteries. Femoralarteries were isolated and balloon injured as described in Example 1.During paired local administration of polythiolated S-nitroso-albuminand polythiolated albumin, [¹¹¹ In]-labeled platelets were administeredintravenously and allowed to circulate after flow was reestablished inthe treated arteries. [¹¹¹ In]-labeled platelet binding was determinedby quantification of radioactivity after flow was re-established for aperiod of 15 minutes. Non-specific [¹¹¹ In]-labeled platelet binding(Uninjured Carotid Artery) was determined from uninjured carotid arteryharvested with femoral arteries. Data are presented as mean +/- SEM pergram of wet tissue weight and are derived from six animals. *P<0.05,PS-BSA vs. pS-NO-BSA.

FIGS. 5A-5B are plots demonstrating the effect of polythiolatedS-nitroso-albumin (pS-NO-BSA) and polythiolated albumin (pS-BSA) onneointimal proliferation 14 days after balloon injury of rabbit femoralartery. Femoral arteries were isolated and balloon injured as describedbelow. pS-BSA or pS-NO-BSA were applied in a paired fashion directlyinto the arterial lumen for 15 minutes and then blood flow wasre-established. After 14 days, arteries were harvested, perfusion-fixed,stained, and subjected to morphometric analysis of intimal and medialareas. Neointimal proliferation is reported as the absolute neointimalarea in FIG. 5A and as a ratio of neointima/media in FIG. 5B in 4-6segments from each artery. Data are expressed as mean +/- SEM and arederived from 15 vessels in the BSA and S-NO-BSA groups, 11 vessels inthe PS-BSA group, 7 vessels in the PS-NO-BSA and SHAM groups, and 5 inthe SNP group. *P<0.05, pS-NO-BSA vs. PS-BSA. +/+P<0.05 Sodiumnitroprusside vs. pS-BSA for both FIGS. 5A and 5B.

FIGS. 6A-6B are plots demonstrating the relationship between neointimalproliferation and the quantity of displaceable NO in preparations ofS-nitrosylated albumin. Femoral arteries were isolated and ballooninjured as described with reference to FIG. 5. Vessels were exposed todifferent preparations of S-nitrosylated albumin with differentdisplaceable NO contents. After 14 days vessels were harvested andanalyzed as described in FIG. 5. Data are expressed as mean +/- SEM andare derived from 7-15 animals in each group. P<0.001 for trend.

FIG. 7 is a plot demonstrating the effect of polythiolatedS-nitroso-albumin (pS-NO-BSA)- and polythiolated albumin(pS-BSA)-treated vessels on platelet cyclic 5'-3' guanosinemonophosphate (cGMP). Rabbit femoral arteries were isolated andballoon-injured as described with reference to FIG. 4. After pairedlocal administration of polythiolated S-nitroso-albumin andpolythiolated albumin for 15 minutes, the vessels were harvested anddivided into 2 mm rings. The rings were then immersed in 100 μl ofplatelet-rich plasma containing 10 μM 3-isobutyl-1-methylxanthine andwere incubated for 1 minute ex-vivo. An equal volume of ice-cold 10%trichloroacetic acid was added to each aliquot and the sample vortexed.Platelet cGMP assay was then performed as described in "Methods." Dataare expressed as mean +/- SEM. *P<0.05.

FIG. 8 is a flow chart illustrating the protocol of Example 4 whichmeasured the effect on balloon-induced injury of pS-NO-BSA or pS-BSA inporcine coronary artery.

FIG. 9 is a histogram which illustrates the diameter (mm) of theneointimal lumen of 14 normocholesterolemic pigs were subjected to aballoon angioplasty which induced injury of the right coronary artery.Thereafter, they received 1.5 μM pS-NO-BSA or pS-BSA as a control.

FIG. 10 is a histogram which illustrates a degree of coronary stenosisobserved at four weeks after angioplasty in pigs which received 1.5 μMpS-NO-BSA or pS-BSA as a control.

FIG. 11. is a histogram which illustrates the extent of coronary spasminduced distal the site of injury as compared to the pre-existing baseline in pigs which received 1.5 μM pS-NO-BSA or pS-BSA as a control.

FIG. 12 is a histogram which illustrates the inner-diameter of the lumenof the right coronary artery of pigs four weeks after they received 1.5μM pS-NO-BSA or pS-BSA as a control.

The invention will now be described in more detail with respect tonumerous embodiments and examples in support thereof.

The term "artificial surface" refers to any synthetic material containedin a device or apparatus that is in contact with blood, blood productsor components, vasculature, or other tissue.

The term "platelet adhesion" refers to the contact of a platelet with aforeign surface, e.g. collagen, artificial surface or device.

The term "platelet aggregation" refers to the adhesion of one or moreplatelets to each other. Platelet aggregation is commonly referred to inthe context of generalized atherosclerosis, not with respect to plateletadhesion on vasculature damaged as a result of physical insult during amedical procedure.

The term "restenosis" refers to the recurrent narrowing of a bloodvessel, usually several months after an injurious insult and as a resultof neointimal proliferation.

The term "passivation" refers to the coating of a surface which therebyrenders the surface non-reactive.

The term "reendothelialization" refers to the proliferation, migrationand spreading of endothelial cells over a surface area which is denudedof endothelial cells, e.g., the surface of a damaged blood vessel.

The term "platelet activation" refers either to the change inconformation (shape) of a cell, expression of cell surface proteins(e.g., the IIb/IIIa receptor complex, loss of GPIb surface protein),secretion of platelet derived factors (e.g., serotonin, growth factors).

The term "lower alkyl" as used herein referes to a branched or straightchain alkyl groups comprising one to ten carbon atoms, including methyl,ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl and the like.

The term "alkoxy" as used herein refers to R₂₀ O, wherein R₂₀ is loweralkyl as defined above. Representative examples of alkoxy groups includemethoxy, ethoxy, t-butoxy and the like.

The term "alkoxyalkyl" as used herein refers to an alkoxy group aspreviuosly defined appended to an alkyl group as previously defined.Examples of alkoxyalkyl include, but are not limited to, methoxymethyl,methoxyethy, isopropoxymethyl and the like.

The term "amino" as used herein refers to --NH₂.

The term "dialkylamino" as used herein refers to R₂₂ R₂₃ N-- wherein R₂₂and R₂₃ are independently selected from lower alkyl, for exampledimethylamino, diethylamino, methyl propylamino and the like.

The term "nitro" as used herein refers to the group --NO₂.

The term "nitroso" as used herein refers to the group --NO.

The term "hydroxyl" or "hydroxy" as used herein refers to the group--OH.

The term "cyano" as used herein refers to the group --CN.

The term "carbomoyl" as used herein refers to H₂ N--C(O) O--.

The term N,N-dialkylcarbomoyl as used herein refers to R₂₂ R₂₃N--C(O)O-- wherein R₂₂ and R₂₃ are independently selected from loweralkyl, for example dimethylamino, diethylamino, methyl propylamino, andthe like.

The term N-alkylcarbamoyl as used herein refers to R₂₂ HN--C(O)O--wherein R₂₂ is selected from lower alkyl, for example methylamino,ethylamino, propylamino, and the like.

The term "aryl" as used herein refers to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and thelike. Aryl groups (including bicyclic aryl groups) can be unsubstitutedor substituted with one, two or three substituents independentlyselected from lower alkyl, haloalkyl, alkoxy, amino, alkylamino,dialkylamino, hydroxy, halo, and nitro. In addition, substituted arylgroups include tetrafluorophenyl and pentafluorophenyl.

The term "arylthio" as used herein refers to R₂₄ S-- wherein R₂₄ isselected from aryl.

The term "alkanoyl" as used herein refers to R₂₂ C(O)-- wherein R₂₂ isselected from lower alkyl.

The term "carboxyl" as used herein refers to --COOH.

The term "guanidino" as used herein refers to H₂ N--C(═NH)NH--.

The term "arylakyl" as used herein refers to a lower alkyl radical towhich is appended an aryl group. Representative arylalkyl groups includebenzyl, phenylethyl, hydroxybenzyl, fluorobenzyl, fluorophenylethyl andthe like.

The term "cycloalkyl" as used herein refers to an alicyclic groupcomprising from 3 to 7 carbon atoms including, but not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term "halogen" or "halo" as used herein refers to I, Br, Cl, or F.The term "haloalkyl" as used herein refers to a lower alkyl radical, asdefined above, bearing at least one halogen substituent, for example,chloromethyl, fluoroethyl or trifluoromethyl and the like.

The term "heteroaryl" as used herein refers to a mono-bycyclic ringsystem containing one or two aromatic rings and containing at least onenitrogen, oxygen, or sulfur in an aromatic ring. Heteroaryl groups(including bicyclic heteroaryl groups) can be unsubstituted orsubstituted with one, two, or three substituents independently selectedfrom lower alkyl, haloalkyl, alkoxy, amino, alkylamino, dialkylamino,hydroxy, halo and nitro. Examples of heteroaryl groups include notlimited to pyridine, pyrazine, pyrimidine, pyridazine, pyrazole,triazole, thiazole, isothiazole, benzothiazole, benzoxazole,thiadiazole, oxazole, pyrrole, imidazole, and isoxazole.

The term "heterocyclic ring" refers to any 3-, 4-, 5-, 6-, or 7-memberednonaromatic ring containing at least one nitrogen atom which is bondedto an atom which is not part of the heterocyclic ring. In addition, theheterocyclic ring may also contain a one additional heteroatom which maybe nitrogen, oxygen, or sulfur.

Compounds of the invention which have one or more asymmetric carbonatoms may exist as the optically pure enantiomers, pure diastereomers,mixtures of enantiomers, mixtures of diastereomers, racemic mixtures ofenantiomers, diastereomeric racemates or mixtures of diastereomericracemates. It is to be understood that the present invention anticipatesand includes within its scope.

As mentioned above, the medical device or instrument may be made, suchthat at least in those portions of it which come into contact withblood, blood components or products, or vascular tissue, include anitric oxide adduct. The nitric oxide adduct can directly or indirectlybe linked to a synthetic material from which all or a portion of thedevice is formed. As representative examples, there may be mentioned:nylon, polyethylene perthalate (Dacron), polytetrafluoroethylene(Gortex).

In another embodiment mentioned above, the nitric oxide adduct can beincorporated into a natural or synthetic matrix which is then used tocoat those same contact surfaces of the device. The matrix can be aliquid into which the nitric oxide adduct has been mixed, which is thencoated onto the contact surfaces of the medical device or instrument andthen allowed to "set", dry, polymerize or otherwise become solid orsemisolid. Examples of such matrix materials include gel-formingmaterials such as are commonly used including hydrogels and starch-basedsemi-solid emulsions and dispersions.

The materials can also be polymers or mixtures of polymers such aspolylactic acid/polylysine copolymer. Alternatively, the matrix can be anatural or synthetic fibrous matrix which is impregnated with a liquidcontaining the nitric oxide adducts either before or after being appliedto the artificial contact surface. Examples of such natural fibrousmatrix materials primarily include filter paper. Examples of suchsynthetic fibrous matrix materials include three-dimensional lattices ofsynthetic polymers and copolymers.

The matrix can also be a material such as nylon or plastic, such aspolystyrene, that is directly or indirectly, i.e., through a linkinggroup, derivitized with the nitric oxide adduct.

As mentioned above the nitric oxide adduct is specifically intended tobe delivered locally at the site of contact of the device or instrumentwith the blood, blood product or component, or vasculature, but need notbe physically associated with the device or instrument. For example, thenitric oxide adduct can be separately administered in a physiologicallyavailable form as a pharmaceutical preparation in a pharmaceuticallyacceptable carrier, such as are described in more detail below. This canbe done by administration during or shortly before the contact orintervention. Where the device is, for example, a catheter, such as acardiac catheter, the nitric oxide adduct preparation can beadministered by injection into the lumen of the catheter.

Compounds contemplated for use in the invention are nitric oxide andcompounds that release nitric oxide or otherwise directly or indirectlydeliver or transfer nitric oxide to a site of its activity, such as on acell membrane, in vivo. As used here, the term "nitric oxide"encompasses uncharged nitric oxide(NO•) and charged nitric oxidespecies, particularly including nitrosonium ion(NO⁺) and nitroxylion(NO⁻). The reactive form of nitric oxide can be provided by gaseousnitric oxide. The nitric oxide releasing, delivering or transferringcompounds, having the structure X-NO wherein X is a nitric oxidereleasing, delivering or transferring moiety, include any and all suchcompounds which provide nitric oxide to its intended site of action in aform active for their intended purpose. As used here, the term "nitricoxide adducts" encompasses any of such nitric oxide releasing,delivering or transferring compounds, including, for example,S-nitrosothiols, S-nitroso amino acids, S-nitroso-polypeptides, andnitrosoamines. It is contemplated that any or all of these "nitric oxideadducts" can be mono- or poly-nitrosylated at a variety of naturallysusceptible or artificially provided binding sites for nitric oxide.

One group of such nitric oxide adducts is the S-nitrosothiols, which arecompounds that include at least one --S--NO group. Such compoundsinclude S-nitroso-polypeptides (the term "polypeptide" includes proteinsand also polyamino acids that do not possess an ascertained biologicalfunction, and derivatives thereof); S-nitrosylated amino acids(includingnatural and synthetic amino acids and their stereoisomers and racemicmixtures and derivatives thereof); S-nitrosated sugars,S-nitrosated-modified and unmodified oligonucleotides (preferably of atleast 5, and more particularly 5-200, nucleotides); and an S-nitrosatedhydrocarbon where the hydrocarbon can be a branched or unbranched, andsaturated or unsaturated aliphatic hydrocarbon, or an aromatichydrocarbon; S-nitroso hydrocarbons having one or more substituentgroups in addition to the S-nitroso group; and heterocyclic compounds.S-nitrosothiols and the methods for preparing them are described in U.S.patent application Ser. No. 07/943,834, filed Sep. 14, 1992, Oae et al.,Org. Prep. Proc. Int., 15(3):165-198, 1983; Loscalzo et al., J.Pharmacol. Exp. Ther., 249(3):726729, 1989, and Kowaluk et al., J.Pharmacol. Exp. Ther., 256:1256-1264, 1990, all of which areincorporated in their entirety by reference.

One particularly preferred embodiment of this aspect relates toS-nitroso amino acids where the nitroso group is linked to a sulfurgroup of a sulfur-containing amino acid or derivative thereof. Forexample, such compounds include the following:S-nitroso-N-acetylcysteine, S-nitroso-captopril, S-nitroso-homocysteine,S-nitroso-cysteine and S-nitroso-glutathione.

Suitable S-nitrosylated proteins include thiol-containing proteins(wherethe NO group is attached to one or more sulfur group on an amino acid oramino acid derivative thereof) from various functional classes includingenzymes, such as tissue-type plasminogen activator(TPA) and cathepsin B;transport proteins, such as lipoproteins, heme proteins such ashemoglobin and serum albumin; and biologically protective proteins, suchas the immunoglobulins and the cytokines. Such nitrosylated proteins aredescribed in PCT Publ. Applic. No. WO 93/09806, published May 27, 1993.Examples include polynitrosylated albumin where multiple thiol or othernucleophilic centers in the protein are modified.

Further examples of suitable S-nitrosothiols include those having thestructures:

    CH.sub.3 (CH.sub.2).sub.x SNO                              (i)

wherein x equals 2 to 20;

    HS(CH.sub.2).sub.x SNO                                     (ii)

wherein x equals 2 to 20; and

    ONS(CH.sub.2).sub.x Y                                      (iii)

wherein x equals 2 to 20 and Y is selected from the group consisting ofhalo, alkoxy, cyano, carboxamido, cycloalkyl, arylalkoxy, loweralkylsulfinyl, arylthio, alkylamino, dialkylamino, hydroxy, carbamoyl,N-alkylcarbamoyl, N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl,hydrogen, nitro and aryl.

Other suitable S-nitrosothiols that are S-nitroso-angiotensin convertingenzyme inhibitors (hereinafter referred to as S-nitroso-ACE inhibitors)are described in Loscalzo, U.S. Pat. No. 5,002,964 (1991) and Loscalzoet al., U.S. Pat. No. 5,025,001 (1991) both of which are incorporated intheir entirety by reference. Examples of such S-nitroso-ACE inhibitorsinclude compounds having structure (1): ##STR1## wherein R is hydroxy,NH₂, NHR⁴, NR⁴ R⁵, or lower alkoxy, wherein R⁴ and R⁵ are lower alkyl,or aryl, or arylalkyl;

R¹ is hydrogen, lower alkyl, arylalkyl, amino, guanidino, NHR⁶, N1R⁶ R⁷,wherein R⁶ and R⁷ are methyl or alkanoyl;

R² is hydrogen, hydroxy, C₁ -C₄ alkoxy, phenoxy, or lower alkyl;

R³ is hydrogen, lower alkyl or arylalkyl;

m is 1 to 3; and

n is 0 to 2.

Other suitable S-nitroso-ACE inhibitors includeN-acetyl-S-nitroso-D-cysteinyl-L-proline,N-acetyl-S-nitroso-D,L-cysteinyl-L-proline,1-[4-amino-2-(S-nitroso)mercaptomethyl butanoyl]-L-proline,1-[2-hexanoyl]-L-proline,1-[5-guanidino-2-(S-nitroso)mercaptomethyl-pentanoyl]-L-proline,1-[5-amino-2-(S-nitroso) mercaptomethyl-pentanoyl]-4-hydroxy-L-proline,1-[5-guanidino-2-(S-nitroso)mercaptomethyl-pentanoyl]-4-hydroxy-L-proline,1-[2-aminomethyl-3(S-nitroso)-mercaptomethyl-pentanoyl-L-proline, andS-nitroso-L-cysteinyl-L-proline.

Additional suitable S-nitroso-ACE inhibitors include those havingstructures (2-3): ##STR2## wherein X is oxygen or sulfur;

--A₁, --A₂ -- is CH--NH or --C═N--; ##STR3## R is selected fromhydrogen, lower alkyl, arylalkyl, and salt forming ion;

R₄ and R₅ are independently selected from hydrogen, halogen, loweralkyl, lower alkoxy, halo substituted lower alkyl, nitro, and SO₂ NH₂ ;##STR4## R₆ is hydrogen, lower alkyl, halo substituted lower alkyl,hydroxy substituted lower alkyl, --(CH₂)_(q) --N (lower alkyl)₂ or--(CH₂)_(q) --NH₂ and q is one, two, three or four; and ##STR5## whereinR⁷ is hydrogen, lower alkyl, alkoxy, halogen or hydroxy and g is asdefined above.

Additional suitable compounds include those having structures (4-11):##STR6##

The S-nitroso-ACE inhibitors can be prepared by various methods ofsynthesis. In general, the thiol precursor is prepared first, thenconverted to the S-nitrosothiol derivative by nitrosation of the thiolgroup with NaNO₂ under acidic conditions (pH=1 to 5) which yields theS-nitroso derivative. Acids which may be used for this purpose includeaqueous sulfuric, acetic and hydrochloric acids. Thiol precursors areprepared as described in the following: U.S. Pat. Nos. 4,046,889 (1977);4,052,511; 4,053,651; 4,113,751, 4,154,840, 4129,571 (1978), and4,154,960 (1979) to Ondetti et al.; U.S. Pat. No. 4,626,545 (1986) toTaub; and U.S. Pat. Nos. 4,692,458 (1987) and 4,692,459 (1987) to Ryanet al., Quadro, U.S. Pat. No. 4,447,419 (1984); Haugwitz et al.; U.S.Pat. No. 4,681,886 (1987), Bush et al., U.S. Pat. No. 4,568,675 (1986),Bennion et al., U.S. Pat. No. 4,748,160 (1988), Portlock, U.S. Pat. No.4,461,896 (1984), Hoefle et al., European Patent Application PublicationNo. 0 088 341 (1983), Huange et al., U.S. Pat. No. 4,585,758 (1986),European Patent application Publication No. 0 237 239, European Patentapplication Publication No. 0 174 162, published in 1986, EuropeanPatent application Publication No. 0 257 485, published in 1988, all ofwhich are incorporated by reference herein.

Another group of such NO adducts are compounds that include at least one--O--NO group. Such compounds include O-nitroso-polypeptides(the term"polypeptide" includes proteins and also polyamino acids that do notpossess an ascertained biological function, and derivatives thereof);O-nitrosylated amino acids (including natural and synthetic amino acidsand their stereoisomers and racemic mixtures and derivatives thereof);O-nitrosated sugars; O-nitrosated-modified and unmodifiedoligonucleotides (preferably of at least 5, and more particularly 5-200,nucleotides); and an O-nitrosated hydrocarbon where the hydrocarbon canbe a branched or unbranched, saturated or unsaturated aliphatichydrocarbon, or an aromatic hydrocarbon; O-nitroso hydrocarbons havingone or more substituent groups in addition to the O-nitroso group; andheterocyclic compounds.

Another group of such NO adducts is the nitrites which have an --O--NOgroup wherein R is a protein, polypeptide, amino acid, branched orunbranched and saturated or unsaturated alkyl, aryl or a heterocyclic. Apreferred example is the nitosylated form of isosorbide. Compounds inthis group form S-nitrosothiol intermediates in vivo in the recipienthuman or other animal to be treated and can therefore include anystructurally analogous precursor R--O--NO of the S-nitrosothiolsdescribed above.

Another group of such NO adducts is the N-nitrosoamines, which arecompounds that include at least one --N--NO group. Such compoundsinclude N-nitroso-polypeptides (the term "polypeptide" includes proteinsand also polyamino acids that do not possess an ascertained biologicalfunction, and derivatives thereof); N-nitrosylated amino acids(including natural and synthetic amino acids and their stereoisomers andracemic mixtures); N-nitrosated sugars; N-nitrosated-modified andunmodified oligonucleotides (preferably of at least 5, and moreparticularly 5-200, nucleotides); and an N-nitrosated hydrocarbon wherethe hydrocarbon can be a branched or unbranched, and saturated orunsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; N-nitrosohydrocarbons having one or more substituent groups in addition to theN-nitroso group; and heterocyclic compounds.

Another group of such NO adducts is the C-nitroso compounds that includeat least one --C--NO group. Such compounds includeC-nitroso-polypeptides (the term "polypeptide" includes proteins andalso polyamino acids that do not possess an ascertained biologicalfunction, and derivatives thereof); C-nitrosylated amino acids(including natural and synthetic amino acids and their stereoisomers andracemic mixtures); C-nitrosated sugars; C-nitrosated-modified andunmodified oligonucleotides (preferably of at least 5, and moreparticularly 5-200, nucleotides); and a C-nitrosated hydrocarbon wherethe hydrocarbon can be a branched or unbranched, and saturated orunsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; C-nitrosohydrocarbons having one or more substituent groups in addition to theC-nitroso group; and heterocyclic compounds.

Another group of such NO adducts is the nitrates which have at least one--O--NO₂ group. Such compounds include polypeptides (the term"polypeptide" includes proteins and also polyamino acids that do notpossess an ascertained biological function, and derivatives thereof);amino acids (including natural and synthetic amino acids and theirstereoisomers and racemic mixtures and derivatives thereof); sugars;modified and unmodified oligonucleotides (preferably of at least 5, andmore particularly 5-200, nucleotides); and a hydrocarbon where thehydrocarbon can be a branched or unbranched, and saturated orunsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon;hydrocarbons having one or more substituent groups; and heterocycliccompounds. A preferred example is nitroglycerin.

Another group of such NO adducts is the nitroso-metal compounds whichhave the structure (R)_(n) --A--M--(NO)_(x). R includes polypeptides(the term "polypeptide" includes proteins and also polyamino acids thatdo not possess an ascertained biological function, and derivativesthereof); amino acids (including natural and synthetic amino acids andtheir stereoisomers and racemic mixtures and derivatives thereof);sugars; modified and unmodified oligonucleotides (preferably of at least5, and more particularly 5-200, nucleotides); and a hydrocarbon wherethe hydrocarbon can be a branched or unbranched, and saturated orunsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon;hydrocarbons having one or more substituent groups in addition to theA-nitroso group; and heterocyclic compounds. A is S, O, or N, n and xare each integers independently selected from 1, 2 and 3, and M is ametal, preferably a transition metal. Preferred metals include iron,copper, manganese, cobalt, selenium and luthidium. Also contemplated areN-nitrosylated metal centers such as nitroprusside.

Another group of such NO adducts is the N-oxo-N-nitrosoamines which havean R--N(O⁻ M⁺)--NO group or an R--NO--NO-- group. R includespolypeptides (the term "polypeptide" includes proteins and alsopolyamino acids that do not possess an ascertained biological function,and derivatives thereof); amino acids(including natural and syntheticamino acids and their stereoisomers and racemic mixtures and derivativesthereof); sugars; modified and unmodified oligonucleotides (preferablyof at least 5, and more particularly 5-200, nucleotides); and ahydrocarbon where the hydrocarbon can be a branched or unbranched, andsaturated or unsaturated aliphatic hydrocarbon, or an aromatichydrocarbon; hydrocarbons having one or more substituent groups; andheterocyclic compounds. R is preferably a nucleophilic (basic) moiety.M+ is a metal cation, such as, for example, a Group I metal cation.

Another group of such NO adducts is the thionitrates which have thestructure R--(S)_(x) --NO wherein x is an integer of at least 2. R is asdescribed above for the S-nitrosothiols. Preferred are the dithiolswherein x is 2. Particularly preferred are those compounds where R is apolypeptide or hydrocarbon and a pair or pairs of thiols aresufficiently structurally proximate, i.e. vicinal, that the pair ofthiols will be reduced to a disulfide. Those compounds which formdisulfide species release nitroxyl ion(NO⁻) and uncharged nitricoxide(NO•). Those compounds where the thiol groups are not sufficientlyclose to form disulfide bridges generally only provide nitric oxide asthe NO⁻ form not as the uncharged NO• form.

Coating of a surface of a medical device with the nitric oxide adductcomprises contacting the surface with the adduct so as to cause thesurface to be coated with the particular adduct. Coating of theartificial surface may be accomplished using the methods described inExample 1, infra, or other standard methods well known to those ofordinary skill in the art. For example, coating a surface with nitricoxide adducts can be achieved by bathing the artificial surface, eitherby itself or within a device, in a solution containing the nitric oxideadduct. In addition, synthetic nitric oxide adducts may be coated ontoan artificial surface by a variety of chemical techniques which are wellknown in the art. Such techniques include attaching the adduct to anucleophilic center, metal, epoxide, lactone, an alpha- orbeta-saturated carbon chain, alkyl halide, carbonyl group, or Schiffbase, by way of the free thiol.

In order to optimize the coating techniques further, standard methodsmay be used to determine the amount of platelet deposition on a sampleof the treated artificial surface. Such methods include the use of ⁵¹Cr-labeled platelets or Indium¹¹¹ -labeled platelets. Other well knowntechniques for evaluating platelet deposition on artificial surfaces aredescribed in Forbes et al. (1978), and Salzman et al. (1981).

It is also contemplated that artificial surfaces will vary depending onthe nature of the surface, and such characteristics as contour,crystallinity, hydrophobicity, hydrophilicity, capacity for hydrogenbonding, and flexibility of the molecular backbone and polymers.Therefore, using routine methods, one of ordinary skill will be able tocustomize the coating technique by adjusting such parameters as theamount of adduct, length of treatment, temperature, diluents, andstorage conditions, in order to provide optimal coating of eachparticular type of surface.

After the device or artificial material has been coated with the nitricoxide adduct, it will be suitable for its intended use, for example,implantation as a heart valve, insertion as a catheter, or forcardiopulmonary oxygenation or hemodialysis. The coated device orartificial surface will be suitable for use in conjunction with ananimal, preferably mammals, including humans.

Another embodiment of a nitric oxide adduct pertains to thederivatization of synthetically derived polymeric materials. Nitricoxide adducts of the formula IA wherein b is an integer from 270 to 500,c is an interger of 1 to 2, d is an integer from 1 to 6, E is a covalentbond, S, N, O, or C--N--C(O)--R⁰, in which R⁰ is H. lower alkyl,cycloalkyl, aryl, heteroaryl, or heterocyclic ring system may beprepared according to the reaction scheme depicted in FIG. 1A, in whichthe biodegradable poly L-lactic acid/poly-L-lysine copolymer prepared asdescribed by Berrera et al., J. Am. Chem. Soc., 115:11010, 1993) isrepresentative of the synthetic polymeric materials defined above. Theprimary amino groups of the compound of formula 13 are reacted with acompound of formula 14, wherein T is an activated carbonyl-containingsubstituent selected from a group consisting of a mixed anyhdride, athioester, an acid chloride, an isocyanate, or a chloroformate, P¹ is asulfur protecting group, and E and d are defined as above to afford acompound of the formula 15 wherein b, c, E, d, and P¹ are defined asabove. A variety of sulfur protecting groups which are compatible withthis process along with methods for their incorporation and removal aredescribed in T. H. Greene and P. G. M. Wuts, Protective Groups inOrganic Synthesis, 2nd edition, John Wiley & Sons, New York, 1991. Thesulfur protecting groups in the compound of the formula 15 are removedto afford the compound of the formula 16 and the thiol moieties arenitrosated to afford a compound of the formula IA using a suitable mildnitrosating agent such as nitrosyl chloride or nitrosoniumtetrafluoroborate in an inert organic solvent or mixture of inertsolvents such as methylene chloride, chloroform, dimethyforamide (DMF),dimethylsulfoxide (DMSO), ethyl acetate, or acetonitrile. In addition,the nitrosation may be performed in the presence or absence of an aminebase such as pyridine or triethylamine. Alternatively, the nitrosationof the compound of the formula 16 may be performed with nitrous acidgenerated in situ from sodium nitrite and hydrochloric acid in anaqueous or mixed aqueous and organic solvent system to afford a compoundof the formula IA.

Nitric oxide adducts of the formula 1B wherein b is an integer from 270to 500, c is an integer of 1 to 2, D is a thiol containing amino acid orpeptide of 1 to 10 amino acids containing 1 to 10 thiols or a thiolcontaining carboxylic acid containing 1 to 10 thiol groups, a is aninteger from 1 to 10, and b and c are defined as above may be preparedaccording to the reaction scheme depicted in FIG. 1B, in which thebiodegradable poly L-lactic acid/poly-L-lysine copolymer prepared asdescribed by Berrera et al. is representative of the synthetic polymericmaterials defined above. The primary amino groups of the compound of theformula 13 wherein b and c are defined as above may be acylated with acompound of the formula 17 wherein Q is halogen, imidazolyl, ortrihalomethoxy in a suitable inert solvent or mixture of solvents suchas DMSO and methylene chloride to afford a compound of the formula 18.The compound of the formula 18 is then reacted with a compound of theformula 19 wherein D and a are as defined above to afford a compound ofthe formula 20. The compound of the formula 20 is then nitrosated toafford a compound of the formula 1B with a suitable mild nitrosatingagent such as nitrosyl chloride or nitrosonium tetrafluoroborate in aninert organic solvent or mixture of inert solvents such as methylenechloride, chloroform, dimethyforamide (DMF), dimethylsulfoxide (DMSO),ethyl acetate, or acetonitrile. In addition, the nitrosation may beperformed in the presence or absence of an amine base such as pyridineor triethylamine. Alternatively, the nitrosation of the compound of theformula 20 may be performed with nitrous acid generated in situ fromsodium nitrite and hydrochloric acid in an aqueous or mixed aqueous andorganic solvent system to afford a compound of the formula 1B.

Another example of a nitric oxide adduct derived from a syntheticpolymeric material is the modification of the L-cysteine amino acidresidues immobilized to modified surface of poly(ethyleneterephalate)which has been activated by pretreatment with3-aminopropyltriethoxysilane followed by glutaraldehyde as described byBui et al., The Analyst, 118:463 (1993). The cysteine thiols may benitrosated with a suitable nitrosating agent such as nitrous acidgenerated in situ from sodium nitrite and hydrochloric acid in anaqueous or mixed aqueous and organic solvent system or, alternatively,with nitric oxide gas or nitrosyl chloride in a suitable inert solventto afford the polymer containing the nitric oxide adduct.

Yet another embodiment of a nitric oxide adduct pertains to thederivatization of a gold or gold coated surface with a self-assembledmonolayer (SAMS) of an ω-substituted alkanethiolate or mixture ofω-substituted alkanethiolates or ω-substituted alkanethiolates andunsubstituted alkanethiolates. Functionalized surfaces of SAMSterminating in carboxylic acids [Collison et al. Langmuir, 8:1247,1992,; Leggett et al., Langmuir, 9:2356, 1993] or amines [Whitesell etal., Angew. Chem. Int. Ed. Engl., 33:871, 1994] have previously beenprepared. These functionalized SAMS may be further derivatized withorganic groups containing one or more nitric oxide adducts as depictedin FIGS. 2A and 2B.

For example, the carboxylyate groups of the SAMS surface composed of thecompound of the formula 21 wherein e is an interger from 2 to 20 may beconverted to an activated acylating species such as the acyl chloridevia reaction with oxalyl chloride and catalytic DMF in an inert solventsuch as THF or chloroform and then the acid chloride may be reacted witha compound of the formula 19 wherein D and a are as defined above toafford a SAMS surface composed of a compound of the formula 22. Thethiol groups of a compound of the formula 22 are nitrosated to afford acompound of the formula IIA with a suitable mild nitrosating agent suchas nitrosyl chloride or nitrosonium tetrafluoroborate in an inertorganic solvent or mixture of inert solvents such as methylene chloride,chloroform, dimethyformamide (DMF), dimethylsulfoxide (DMSO), ethylacetate, or acetonitrile. In addition, the nitrosation may be performedin the presence or absence of an amine base such as pyridine ortriethylamine. Alternatively, the nitrosation of the compound of theformula 22 may be performed with nitrous acid generated in situ fromsodium nitrite and hydrochloric acid in an aqueous or mixed aqueous andorganic solvent system to afford a compound of the formula IIA.

As yet another example, the amine groups of the SAMS surface composed ofthe compound of the formula 23 wherein e is an interger from 2 to 20 maybe reacted with a compound of the formula 14 wherein T, E, d and P¹ aredefined as above to afford a SAMS surface composed of a compound of theformula 24. After deprotection of the thiol moieties of the compound ofthe formula 24, the free thiol groups are nitrosated to afford acompound of the formula IIB using a suitable mild nitrosating agent suchas nitrosyl chloride or nitrosonium tetrafluoroborate in an inertorganic solvent or mixture of inert solvents such as methylenecholoride, chloroform, dimethyformamide (DMF), dimethylsulfoxide (DMSO),ethyl acetate, or acetonitrile. In addition, the nitrosation may beperformed in the presence or absence of an amine base such as pyridineor triethylamine. Alternatively, the nitrosation of the free thiolgroups may be performed with nitrous acid generated in situ from sodiumnitrite and hydrochloric acid in an aqueous or mixed aqueous and organicsolvent system to afford a compound of the formula IIB.

Particularly preferred nitric oxide adducts are polynitrosylatedpeptides and proteins. Synthesis of polynitrosated peptides and proteinscan be achieved in several ways. 1) Mono S-nitrosylation is bestachieved by incubating peptides and proteins (in deionized water in anequimolar concentration of acidified nitrite (final concentration 0.5 NHCl) for a period of 1-30 minutes. The incubation time depends on theefficiency of nitrosation and the tolerance of the protein. Nitrosationcan also be achieved with a variety of other nitrosating agentsincluding compounds such as S-nitroso-cystein, S-nitrosoglutathione andrelated alkyl nitrites. These compounds are to be used when the peptideor protein does not tolerate harsh acidic conditions, e.g. humanhemoglobin.

There are two principal ways of achieving poly S-nitrosation. In thefirst, the peptide or protein is reduced in 100-1000 molar excessdithiothreitol for 30-60 minutes. This exposes intramolecular thiols.The peptide or protein is separated from dithiothreitol by gelfiltration (G-25). The protein is then exposed to increasingconcentrations of acidified nitrite (0.5 N HCl) in relative excess overprotein. Complementary measurements of Saville indicate whenS-nitrosation is complete. For example, with albumin, this procedureleads to approximately 20 intramolecular S-NO derivatives.

Alternatively, the protein can be treated with thiolating agent such ashomocysteine thiolactone. This tends to add homocystine groups toexposed amine residues in proteins. The derivatized protein can then beS-nitrosated by exposure to acidified nitrite. Exposure to increasingconcentrations of nitrite with complementary measurements of Saville canbe used to ascertain when S-nitrosation is maximal. Alternatively, thiolgroups can be quantified on the protein using standard methodologies andthen the protein treated with a stoichiometric concentration ofacidified nitrite (0.5 N HCl).

Polynitrosation of nucleophilic functional groups (other than thiol) canbe achieved when proteins are incubated with excess acidified nitrite.The order of protein reactivity is tyrosine followed by amines onresidues such as trytophan. Amide linkages are probably less reactive.Accordingly, many NO groups can be added to proteins by simplyincubating the protein with high excess acidified nitrite. For example,exposure of albumin to 1000 fold excess nitrite leads to approximately200 moles of NO/mole protein. These experiments are performed in 0.5normal HCl with incubations for approximately one hour. ¹⁵ N NMR can beused to determine where the addition (or substitution) by NO takesplace.

Finally, nitrosation can be achieved by exposure to authentic nitricoxide gas under anaerobic conditions. For successful nitrosationproteins should be incubated in at least 5 atmospheres of NO gas forseveral hours. Incubation time is protein specific. This can lead to NOattachment to a variety of protein bases. Best characterized reactionsinvolve primary amines. This mechanism provides a pathway to sustainN-nitrosation reactions without deamination. Specifically, exposure toacidified nitrite would otherwise lead to deamination of primary amineswhereas this method leads to formation of N-hydroxynitrosamines withpotent bioactivity. Similar substitutions at other basic centers alsooccur.

The method of the invention provides significant advantages over currentattempts to reduce platelet deposition on artificial surfaces. Asdemonstrated by the inventors, a surface can be coated with nitric oxideadducts using simple, effective methods. The coated surfaces may be usedimmediately, or stored and used at a later date. In addition, by coatingthe surface itself, this method eliminates the need for systemicadministration of anti-thrombogenic agents which are often ineffective,have serious adverse side effects, or are unsuitable for use in certainpatients. Also, the inhibition of platelet deposition provided by theinvention is completely and immediately reversible, a need which isespecially important in patients with cardiac or vascular disease.

By preventing platelet deposition or thrombus formation, the inventionis also useful in preventing serious vascular complications associatedwith the use of medical devices. These complications occur as a resultof increased platelet deposition, activation, and thrombus formation orconsumption of platelets and coagulation proteins. Such complicationsare well known to those of ordinary skill in the medical arts andinclude myocardial infarction, pulmonary thromboembolism, cerebralthromboembolism, thrombophlebitis, thrombocytopenia, bleeding disordersand any additional complication which occurs either directly orindirectly as a result of the foregoing disorders.

In another embodiment, the invention relates to a method for preventingthe deposition of platelets on a surface comprising contacting thesurface with a nitric oxide adduct in combination with at least oneadditional anti-thrombogenic agent. The term "anti-thrombogenic" agentrefers to any compound which alters platelet function, or interfereswith other mechanisms involved in blood clotting, such as fibrinformation. Examples of such compounds include, but are not limited to,heparin, warfarin, aspirin, indomethacin, dipyridamole prostacyclin,prostaglandin-E₁ or sulfinpyrazone.

This method for coating a surface with a nitric oxide adduct incombination with another anti-thrombogenic agent will be accomplishedusing the methods described previously for coating a surface with anitric oxide adduct alone, and are suitable for any and all types ofnatural tissue and artificial surfaces. The appropriate coatingconcentration of the other anti-thrombogenic compound is determinedusing routine methods similar to those described previously. The coatedsurfaces may be used in the same manner described for those surfacescoated with nitric oxide adducts alone.

By coating a surface with a nitric oxide adduct in combination with atleast one other anti-thrombogenic agent, one will be able to not onlyprevent platelet deposition, which is the initial event in thrombusformation, but also to limit fibrin formation directly, by inhibitingfactor VIII, and platelet granule secretion, and indirectly, byinhibiting plasminogen activator inhibitor (PAI-1) release fromplatelets. Thus, by coating a surface with agents that both preventplatelet deposition and interfere with other platelet functions whichcontribute to coagulation, the invention provides a further means forpreventing thrombus formation.

In a further embodiment, the invention relates to a method forpreventing thrombus formation on a damaged vascular surface in ananimal, comprising applying a nitric oxide adduct directly to thedamaged surface. The term "damaged vascular surface" refers to anyportion of the interior surface of a blood vessel in which damage to theendothelium or subendothelium, narrowing or stenosis of the vessel hasoccurred. The invention is especially suitable for use in coronaryarteries, but is beneficial in other damaged arteries and also in veinsincluding particularly those used in arterial or venous bypassreplacement where they are susceptible to damage from the typicallyhigher arterial pressures to which they are unaccustomed.

The nitric oxide adduct is applied directly to the damaged vascularsurface by using an intraarterial or intravenous catheter, suitable fordelivery of the adduct to the desired location. The location of damagedarterial surfaces is determined by conventional diagnostic methods, suchas X-ray angiography, performed using routine and well-known methodsavailable to those of skill within the medical arts. In addition,administration of the nitric oxide adduct using an intraarterial orintravenous catheter is performed using routine methods well known tothose in the art. Typically, the preparation is delivered to the site ofangioplasty through the same catheter used for the primary procedure,usually introduced to the carotid or coronary artery at the time ofangioplasty balloon inflation.

The compounds of this invention can be employed in combination withconventional excipients, i. e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral application whichdo not deleteriously react with the active compounds. Suitablepharmaceutically acceptable carriers include, but are not limited to,water, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethylcellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringand/or aromatic substances and the like which do not deleteriously reactwith the active compounds. For parenteral application, particularlysuitable vehicles consist of solutions preferably oily or aqueoussolutions, as well as suspensions, emulsions, or implants. Aqueoussuspensions may contain substances which increase the viscosity of thesuspension and include, for example, sodium carboxymethyl cellulose,sorbitol, and/or dextran. Optionally, the suspension may also containstabilizers.

The term "therapeutically effective amount," for the purposes of theinvention, refers to the amount of the nitric oxide adduct which iseffective to achieve its intended purpose. While individual needs vary,determination of optimal ranges for effective amounts of each nitricoxide adduct is within the skill of the art. Generally, the dosagerequired to provide an effective amount of the composition, and whichcan be adjusted by one of ordinary skill in the art will vary, dependingon the age, health physical condition, sex, weight, extent of disease ofthe recipient, frequency of treatment and the nature and scope of thedesired effect. The preparations, which are suitable for treatment ofartificial surfaces, such as of a medical device, and endothelium areused in concentrations of about 500-700 mM of adduct delivered by dripinfusion sterile in a physiological liquid over 2-3 minute periods inamounts of 2-3 ml per 25 kg body weight.

As demonstrated by the inventors, direct application of a nitric oxideadduct to a damaged vascular surface, coats the surface, therebydecreasing the thrombogenicity of the surface. As further demonstratedby the inventors, local application of the nitric oxide adduct to thedamaged vascular surface can be accomplished at doses much lower thanthose required to exert a systemic effect. Thus, this method provides asignificant and an unexpected advantage over the use of systemicanti-thrombogenic agents to prevent thrombus formation in damagedvessels.

EXAMPLE 1 NO Adducts Make Artificial Surfaces Less Thrombogenic

One of the best ways to demonstrate that an artificial surface exposedto blood has been made less thrombogenic is to measure or quantitate thenumber of blood platelets that collect on that surface. This methodrequires the removal of platelets from an animal or human subject. Theplatelets are labeled with a radioactive material such as Indium¹¹¹,which emits gamma rays, detectable by a gamma counter placed 3 to 6inches away from the source of radioactive platelets. The labeledplatelets are either reinjected into the animal or human in vivo, orcontacted with the artificial surface in vivo. Platelets will adhere toartificial surfaces or acutely damaged arterial surfaces. Thus, thenumber of normal platelets and radioactive platelets which stick to thesurface is an indication of the thrombogenicity of the surface.

The inventors have used this methodology in experiments to demonstratethat nitric oxide adducts decrease the thrombogenicity of an artificialsurface or a damaged natural arterial surface. The following experimentsdemonstrate that coating artificial surfaces, such as synthetic vasculargraft material, with a nitric oxide adduct, decreases plateletdeposition and makes the surface significantly less thrombogenic thanpreviously used agents such as albumin alone. In addition, theexperiments demonstrate that polyvinyl chloride (PVC) tubing, which isused extensively in artificial kidney and heart-lung machines, can becoated with an nitric oxide adduct such as S-nitroso-albumin, to make itless thrombogenic.

Protection of Synthetic Vascular Grafts

First, the inventors coated dacron grafts and cardiac catheters withS-nitroso-bovine serum albumin (BSA). In three separate experiments, anidentical pair of 6 mm (internal diameter) knitted dacron grafts, 5 cm.in length, were prepared for surgical placement in the transectedcarotid arteries of three anesthetized dogs. No heparin was given. Onegraft was soaked in 5% BSA and the other graft was soaked in 5% BSAcombined with 0.5 mM nitric oxide (producing S-nitroso-BSA) for one hourprior to insertion, and then rinsed in saline. The grafts were suturedin place with a continuous 6-0 proline suture.

Indium-labeled Platelets

Indium¹¹¹ -labeled platelets are very useful in detecting plateletaccumulation on vascular grafts. Therefore, Indium¹¹¹ -labeled plateletswere prepared according to standard methods described in Heyns "Methodfor Labeling Platelets with In¹¹¹ -oxine". In: Platelet Kinetics andImaging Vol. II, Editors Heyns et al., CRC Press, 1985; and Sheffel etal., J. Nucl. Med., 20: 524-531, 1979, and injected prior to insertionof the grafts. Following graft insertion, the dogs were observed for twohours, then both grafts were removed, rinsed, and weighed. The graftswere then placed in a Nal gamma well counter and counted for fourminutes.

The three grafts coated with BSA alone had an average of654,000+/-89,000 counts/4 minutes. In contrast, the three grafts coatedwith S-nitroso-BSA had an average of 278,000+/57,000 counts/4 minutes(P<0.005). The average percent increase in weight for the three graftsdue to thrombus formation on the luminal surface with BSA alone, was410%+/-97%, while the percent increase in weight for the three graftsincubated with nitroso-BSA was 196%+/-71% (P<0.005).

These data show that during exposure of the graft to circulating bloodover a period of two hours, there was considerably less plateletdeposition and clotting on the synthetic grafts treated withS-nitroso-BSA. Thus the results demonstrate that S-nitroso-BSA coatingof synthetic vascular grafts provides protection against early plateletdeposition.

In addition, three pairs of 5 FR USC1 catheters were studied. Onecatheter was soaked in 5% BSA, while the other catheter was soaked in amixture of S-nitroso BSA for one hour. The catheters were rinsed withsaline and one each was inserted into the right or left femoral arteriesof the dogs described above, and left for two hours. Each catheter wasflushed with normal saline every one-half hour, but no heparin wasgiven. The catheters were then removed and rinsed with saline. Equallengths of the catheters were cut from the distal ends and each one wasplaced in a Nal gamma counter and the radioactivity was counted for fourminutes.

The counts for the three catheters coated with BSA alone had an averagecount of 9,000±1,100. In contrast, the three catheters coated with 5%BSA+0.5 Mm nitric oxide had only 2,850±800 counts. Thus, there weresignificantly fewer platelets deposited on the catheters coated withS-nitroso-BSA, than those coated with BSA alone. These experimentsdemonstrate that synthetic vascular grafts coated with S-nitroso-BSA andimmediately implanted, are significantly less thrombogenic than graftscoated with BSA alone.

The inventors conducted an additional experiment to investigate whetherS-nitroso-BSA can be used to coat a surface such as polyvinyl chloride(PVC), and in addition, whether such surfaces can be treated at onetime, and used at a later time. In this experiment, three pieces of PVC,3 mm in internal diameter and 2 cm. in length were soaked in BSA for 4hours, allowed to dry, and placed in a dark place. Three identicalpieces of PVC tubing were soaked in an S-nitroso-BSA solution for 4hours, dried, and also placed in the dark. The lengths of PVC tubingwere kept in the dark to minimize potential inactivation of the nitricoxide-donating compounds caused by exposure to light.

Three days after coating, a pair of PVC tubing pieces, one coated withBSA, and one coated with S-nitroso-BSA, were placed as a shunt in eachof the two femoral arteries of a dog. The dog was injected withIndium¹¹¹ labeled platelets as previously described. Two hours after thePVC shunts were placed in the circulation with radioactive platelets,they were removed and placed in the Nal gamma counter.

The counts on the BSA coated shunt were 200,870/4 minutes, whereas onthe S-nitroso-BSA coated graft, the counts were only 97,510/4 minutes.Thus, the shunts coated with S-nitroso-BSA have significantly fewerplatelets deposited on its internal surface than the one coated withnitroso-BSA.

EXAMPLE 2 Na Nitroprusside Coated Damaged Arterial Surfaces Are LessThrombogenic

The following experiments demonstrate that nitric oxide-donatingcompounds, such as sodium nitroprusside and S-nitroso-BSA, can beapplied directly to damaged arterial or venous surfaces (blood vessels)to inhibit platelet deposition and thrombus formation.

The inventors developed an animal model which allows them to mimic apatient with narrowing of the coronary or other arteries and arterialdamage caused by atherosclerosis or after angioplasty, atherectomy orother procedure. The model uses anesthetized dogs with open chest andexposed heart. Briefly, an electromagnetic flow probe is placed on thecoronary artery to continuously measure blood flow through the artery.Then the arterial wall is damaged(intima and media) by clamping theartery several times with a surgical clamp. In the area of arterialdamage, a plastic encircling cylinder is placed around the outside ofthe coronary artery to produce a 70% narrowing or reduction in the lumengradually diameter. This mimics atherosclerotic narrowing of arteries inpatients. Platelet-mediated thrombi periodically form in the stenosedlumen, gradually cutting off the coronary blood flow. Subsequently, thethrombi embolize distally and blood flow is restored. This process,which occurs periodically, produces cyclical reductions in flow,hereinafter referred to as "cyclic flow reductions" (CFRs). If no actionis taken to prevent platelet interaction with the damaged arterial wall,these CFRs will continue to occur for many hours.

The inventors have determined that CFRs represent an interaction betweenplatelets and the clotting system, and damaged endothelial cells innarrowed or stenosed arterial walls. In addition, CFRs occur in humanarteries which are narrowed by atherosclerosis, and the resultingperiodic clot formation can cause chest pain or leg pain in patientswith atherosclerotic narrowing of coronary or leg arteries. Finally, theCFRs due to platelet-mediated clotting can be exacerbated by furtherdamage to the arterial wall.

During the course of this study it was observed that when arterial wallwas damaged initially by clamping the artery with a surgical clamp,platelet thrombi formed, and CFRs were produced. As a result of thisobservation, the following experiments were conducted to determine ifdirect infusion of an NO donor such as sodium nitroprusside can make adamaged arterial wall less thrombogenic.

The following experiments demonstrate that nitric oxide-donatingcompounds, such as sodium nitroprusside and S-nitroso-BSA can be applieddirectly to damaged arterial surfaces (blood vessels) to inhibitplatelet deposition and thrombus formation.

In five anesthetized dogs, both carotid arteries were exposed. Two 3 FRUSC1 catheters were prepared for arterial implantation. One catheter wassoaked in a 5% BSA solution for 12 hours, while the other was soaked ina 5% BSA solution which also contained 1 mg/ml of sodium nitroprusside.One each of the two coated catheters was placed randomly in the right orleft carotid artery of the dog through a small incision sealed with a6-0 proline suture. The catheters were advanced for 5 cm into thearterial lumen. The dogs were not given any heparin. The catheters wereremoved 6-8 hours later and examined for clotting on the catheter walland at the site where the catheter entered the carotid wall. There wasconsiderably more clotting on the BSA-coated catheter compared to thecatheter coated with BSA plus sodium nitroprusside.

In five open-chested anesthetized dogs, the coronary artery wasdissected out and instrumented for measuring CFRs as previouslydescribed. The inventors observed that intravenous infusion of sodiumnitroprusside directly into the artery (at a dose of between 4 and 10μg/kg/min. for up to 30 minutes) resulted in a decrease in vivo plateletactivity and CFRs were abolished. In addition, the circulatingnitroprusside appeared to coat the damaged arterial wall, thus making itless thrombogenic. The CFRs were observed to continue until the sodiumnitroprusside infusion had been given for 15 minutes. Then, the CFRsceased, which suggests that the platelets were no longer adhering to thearterial wall. The sodium nitroprusside intravenous infusion was thenstopped. The direct in vivo inhibition of circulating platelets normallystops within 10-15 minutes. However, after the in vivo inhibition of theplatelets by the presence of circulating sodium nitroprusside was gone,the CFRs did not return. This indicates that the previously circulatingsodium nitroprusside left a protective coating on the previously damagedarterial surface. The inventors have termed this protective coatingprocess "passivation."

The inventors then showed that if one gently rolls the artery betweenthe fingers, the CFRs return immediately. This suggests that theprotective coating provided by sodium nitroprusside, has been removedfrom the internal surface of the previously damaged artery, thus,allowing platelets to resume interaction with the unprotected arterialwall and produce CFRs. In order to demonstrate that this was a localphenomenon affecting the damaged artery, and not due to a systemiceffect inhibiting all the circulating platelets, the followingexperiments were performed.

Open-chest anesthetized dogs were studied. In the dog, and also inhumans, the two major branches of the main left coronary artery whichare approximately equal in size, are called the left circumflex (circ)and the left anterior descending (LAD), coronary arteries. In theexperiments, both branches were instrumented with a flow measuringdevice, were given equal arterial wall damage (endothelial and medialdamage), and had encircling plastic cylinders placed on them to produceequal amounts of narrowing or stenosis.

Following the induction of damage in both coronary arterial branches,CFRs were observed in both the LAD branch and the circumflex branches ofthe left coronary artery, indicating that the circulating platelets wereadhering to both the narrowed part of the damaged circumflex artery andalso to the damaged LAD artery. Sodium nitroprusside (10 mg/kg) was theninfused directly into the circumflex coronary artery over 30 seconds.Following the infusion, the CFRs in the circ disappeared while theycontinued in the LAD coronary artery. This demonstrates that the sodiumnitroprusside had a local protective effect on the damaged circ, andthat the dose of sodium nitroprusside was not high enough to affectcirculating platelets or, after recirculation dilution, to protect thedamaged LAD wall.

CFRs due to platelets adhering and aggregating on the damaged arterialwalls were observed in both arteries, each independent of the other.Therefore, by injecting the sodium nitroprusside into the circumflexbranch, the inventors were able to coat this damaged artery directly. Inaddition, the circulating concentration of sodium nitroprussideremaining after local infusion appears to be too low to have a systemiceffect on platelets. Thus, the inventors demonstrated that theprotective effect exerted by localized application of sodiumnitroprusside is a local effect, and can be applied directly to protectparticular segments of a damaged artery.

Experiments identical to those described above were repeated using anitric oxide-bovine serum albumin adduct (BSA-NO) (with approximately0.5 mM NO concentration) given selectively into the circumflex coronaryartery. The inventors show that using BSA-NO as the NO adduct providesbetter passivation and the effect lasts longer. When the protectiveBSA-NO coating has been on the damaged arterial wall for 4 to 5 hours,the BSA can be recharged with new NO molecules by infusing sodiumnitroprusside intravenously (5-10 μg/kg for 20 minutes) or directly intothe coronary artery (10 mg/kg for 30 seconds).

EXAMPLE 3 pS-NO-BSA Treats Vascular Injury

Materials: Sulfanilamide and N-(1-naphthyl) ethylenediaminedihydrochloride were purchased from Aldrich Chemical Co., Milwaukee,Wis. Sodium bicarbonate, sodium chloride, sodium phosphate, sodiumnitrite, potassium phosphate-monobasic, 40% formaldehyde solution andsucrose were purchased from Fischer Scientific, Fairlawn, N.J. SephadexG25 was purchased from Pharmacia, Piscataway, N.J., IODO-BEADS werepurchased from Pierce, Rockford, Ill. and Na[¹²⁵ I] from New EnglandNuclear, Boston, Mass. [¹¹¹ In] oxine was purchased from Amersham,Arlington Heights, Ill. Monoclonal mouse anti-proliferating cell nuclearantigen was purchased from Dako A/S, Denmark. All other chemicals werepurchased from Sigma Chemical Co., St. Louis, Mo.

Citrate-phosphate-dextrose anticoagulant solution (CPD) contained 10 mMcitric acid, 90 mM trisodium citrate, 15 mM NaH₂ PO₄ H₂ O, and 142 mMdextrose, pH 7.35. Tris-buffered saline consisted of 10 mMtris[hydroxymethyl]aminoethane, pH 7.4, and 150 mM NaCl.Acid-citrate-dextrose contained 100 mM trisodium citrate and 142 mMdextrose, pH 6.5. Phosphate-buffered saline contained 10 mM sodiumphosphate and 150 mM NaCl, pH 7.4.

Synthesis of S-nitroso-species: S-NO-BSA was synthesized as previouslydescribed..sup.(10) Fatty acid-free bovine serum albumin (200 mg/ml) wasexposed to a 1.4 molar-fold excess of NaNO₂ in 0.5 N HCl for 30 minutesat room temperature and neutralized with an equal volume of TBS and 0.5N NaOH. Thiolated bovine serum albumin (pS-BSA) was prepared afterBenesch and Benesch..sup.(14) Briefly, essential fatty acid-free bovineserum albumin (50 mg/ml) was dissolved in water withN-acetyl-homocysteine thiolactone (35 mM) and 0.05% polyethylenesorbitanmonolaurate. Equimolar Silver nitrate was slowly added at roomtemperature over 90 minutes at pH 8.5. Excess thiourea (70 mM) was addedand the pH lowered to 2.5. Excess silver nitrate was removed by Dowex 50chromatography with the mobile phase consisting of 1M thiourea, pH 2.5,and excess thiourea was removed by Sephadex G-25 chromatography. pS-BSAwas prepared within two days of nitrosylation and stored at 4° C.Nitrosylation of PS-BSA was accomplished with 3.6 mM NaNO₂ in 0.5 HClfor 30 minutes at room temperature. The solution was adjusted to pH 4.0with 0.5 NAOH after nitrosylation. In platelet binding studies, 0.1 mMEDTA was added to pS-BSA prior to nitrosylation.

The content of S-nitrosothiol was determined by the method of Saville(Wistow et al., J. Nucl. Med. 19:483-487, 1978). Protein content wasdetermined using the method of Lowry and colleagues (Marcus Salier,FASEB.J., 7:516-522, 1993).

Preparation of [¹²⁵ I]-labeled S-NO-BSA and [¹¹¹ In] labeled platelets:BSA (0.1 mg/ml) was combined with two IODO-BEADS and 0.1 mCi of Na[¹²⁵I]. The solution was incubated for 45 minutes and unincorporated Na[¹²⁵I] was removed by gel filtration with Sephadex G25 equilibrated with TBScontaining 0.1 mg/ml BSA. [¹²⁵ I] BSA had a specific activity-of 5.7×10⁶cpm/μg and was S-nitrosylated as described for unlabelled BSA to achievea final specific activity of 4×10⁴ cpm/mg BSA. [¹¹¹ In]-labeling ofplatelets was performed after the method of Wistow andcolleagues..sup.(17)

Animal Preparation: All animal preparations were performed within theinstitutional guidelines of the Brockton/West Roxbury Department ofVeteran Affairs Medical Center and Boston University Medical Center, andin accordance with the guiding principles of the American PhysiologicalSociety. New Zealand white rabbits (3.5-4.2 kg) of either sex werepremedicated with 5 mg/kg intramuscular (IM) xylazine hydrochlolide(Miles Pharmaceuticals, Shawnee Mission, Kans.), and 0.1 mg/kgsubcutaneous (SC) atropine sulfate (Lyphomed, Deerfield, Ill.) fifteenminutes prior to the induction of anesthesia. Anesthesia was inducedwith 40 mg/kg IM ketamine hydrochloride (Fort Dodge Laboratories, FortDodge, Iowa) and 5 mg/kg IM acepromazine maleate (Aveco Company, Inc.,Fort Dodge, Iowa). Additional doses of ketamine hydrochloride wereadministered as necessary to maintain anesthesia. For survival studies,100,000 U penicillin G (Apothecon of Bristol-Myers Squibb, Princeton,N.J.), was administered IM perioperatively. The skin over the femoralarteries was next infiltrated with 1% lidocaine (Astra Pharmaceuticals,Inc., Westborough, Mass.) and the common femoral arteries were exposedfrom the inguinal ligament to the superficial femoral artery. Arterieswere cleared of connective tissue, side branches were ligated, and thesuperficial femoral artery was suspended with silk ties. A 1.5-to-2.0 cmlength of femoral artery was isolated from the circulation proximallyand distally with neurosurgical microaneurysm clips. The superficialfemoral artery was cannulated with a 2F Fogarty balloon catheter(American Edwards Laboratories, Santa Ana, Calif.) that was passed intothe isolated segment of femoral artery. The balloon was inflated withsufficient air to generate slight resistance and withdrawn three times.A 20 g angiocath was then inserted in the arteriotomy and 1 ml of 25.8mg/ml PS-NO-BSA or 49.2 mg/ml S-NO-BSA was administered over 15 minutes.The contralateral femoral artery was prepared identically and anappropriate control (25.8 mg/ml pS-BSA, 49.2 mg/ml BSA or 0.66 mg/mlsodium nitroprusside) was administered. For binding studies, 0.5 ml of[¹²⁵ I]-labeled nitrosylated albumin or control was administered.Following administration of the agent, the superficial femoral arterywas ligated and flow reestablished. Sham-operated animals underwentsurgical exposure and sidebranch ligation, but no balloon injury wasperformed or local delivery administered. The area of balloon injury wasmarked by surgical staples in the adjacent muscle fascia. For chronicstudies, the incision was closed with subcuticular absorbable suture andthe animals allowed to recover. For acute studies, blood was allowed tocirculate through the treated areas for 15 minutes prior to vesselharvest. In some experiments, a distant control vessel, the rightcarotid artery, was isolated and harvested without any othermanipulation.

cGMP analysis: Whole blood was obtained from fasting human volunteersand platelet-rich plasma (PRP) was prepared by centrifugation. Plateletcounts were determined using a Coulter counter model ZM (CoulterDiagnostics, Hileah, FL). After balloon injury and treatment withpS-NO-BSA or PS-BSA, arterial segments were harvested and 2-mm segmentswere incubated with 100 μl of PRP containing 10 μMisobutylmethylxanthine. After 1 minute, an equal volume of ice-cold 10%trichloroacetic acid was added to each aliquot and the sample vortexed.Enzyme-linked immunoassay of cGMP was then performed (Cayman ChemicalCompany, Ann Arbor, Mich.). Separate 2 mm vessel segments were alsoassayed for tissue cGMP after treatment with ice-cold 10%trichloroacetic acid and sonication (Heat Systems-Utrasonics, Inc.,Plainview, N.Y.).

Tissue processing and analysis: On the 14th postoperative day, animalswere euthanized with 120 mg/kg intravenous sodium pentobarbital (AnproPharmaceuticals, Arcadia, Calif.), and the abdominal aorta and inferiorvena cava interrupted by silk ties. A 7F plastic cannula was insertedinto the abdominal aorta and the vessels perfused clear with salinefollowed by fixation at 100 mm Hg pressure with 10% buffered formalin.The vessels were stored in 10% buffered formalin and the samplesparaffin-embedded and microtome-sectioned. Six sections were made alongthe length of each injured segment of vessel and stained with Verhoeff'sstain for elastic tissue. The areas within the lumen, internal elasticmembrane, and external elastic membrane were measured by a blindedobserver using computerized digital planimetry (Zeiss, West Germany).The areas within the lumen, internal elastic membrane and externalelastic membrane were analyzed. Sections with obstructive thrombusimpairing analysis were discarded.

In a separate set of animals, vessels were perfusion-fixed with 10%buffered formalin seven days after injury and processed for analysis ofproliferating cells within 12 hours as described above. Sections werestained for proliferating cell nuclear antigen and adjacent sectionswere stained with hematoxylin and eosin. Five representative sectionsfrom each segment were examined. Total nuclei were counted from thehematoxylin and eosin slides and percent PCNA positive cells weredefined as the number of PCNA-positive nuclei divided by the totalnumber of nuclei multiplied by 100.

[¹¹¹ In]-labeled platelet studies: Animals were prepared and treatedwith pS-NO-BSA or pS-BSA as described above. Five minutes prior to therelease of the vascular clamps, autologous [¹¹¹ In]-labeled plateletswere infused via the femoral vein, and the blood was allowed torecirculate for 15 minutes prior to harvest. Platelet adhesion wasquantified with a gamma counter (Capintec Instruments, Inc., Pittsburgh,Pa.) and normalized to tissue wet weight.

Statistics. Data are presented as mean +/- SEM. Treatments wereadministered in a paired fashion with one femoral artery receivingS-nitrosylated protein and the contralateral artery receiving theappropriate non-nitrosylated control. Sodium nitroprusside was given toa separate set of animals. Data were tested for normality using theKolmogorov-Smirnov algorithm and for equal variance with the LeveneMedian test. Normally distributed variables were compared using thepaired t-test and non-normally distributed variables using the Wilcoxonsign-ranks test or the Mann-Whitney rank-sum test. Non-paired data werecompared using an independent t-test. Statistical analysis ofdose-response was performed by one-way analysis of variance. Statisticalanalysis of dose-response was performed by one-way analysis of variance.Statistical significance was accepted if the null hypothesis wasrejected with P<0.05.

Results

NO content of S-nitrosothiol species: The synthesis of S-NO-BSA resultedin a final protein concentration of 755 μM (49.2 mg/ml) and yielded adisplaceable NO content of 230±60 μM, yielding a stoichiometry of 0.3+/- 0.08 moles NO/mole albumin (n=11). Thiolation and S-nitrosylation ofBSA produced a final protein concentration of 391 μM (25.8 mg/ml, n=8)and yielded a 5.9-fold increase in displaceable NO content with amaximum content of 2300 μM displaceable NO as compared to S-NO-BSA.Local delivery consisted of 1 ml of either S-nitrosylated protein orcontrol solution instilled in the lumen of the femoral artery.

S-NO-BSA binding: The binding of locally and systemically delivered [¹²⁵I]-labeled S-NO-BSA to balloon-injured rabbit femoral artery is shown inFIG. 3. Compared with systemic administration to an injured artery,local delivery of [¹²¹ ]-S-NO-BSA to the site of injury was associatedwith a 26-fold increase in binding (140.4 +/- 3.9×10³ cpm/gm vs. 5.4 +/-0.9×10³ cpm/gm, n=4; P=0.029). Endothelial denudation facilitatedS-NO-BSA binding as systemic administration of [¹²¹ I]-S-NO-BSA resultedin significant deposition at the site of balloon injury compared to anuninjured control vessel exposed to systemically delivered [¹²⁵I]-S-NO-BSA (5.4±0.9×10³ cpm/gm vs. 3.0 +/- 0.3 cpm/gm, n=4; P=0.038).

pS-NO-BSA effect on platelet binding to injured vessel: Since plateletadhesion to the injured arterial surface is important in theproliferative response to injury, we investigated the effects ofpS-NO-BSA on platelet deposition after balloon injury, the results ofwhich are shown in FIG. 4. The local administration of pS-NO-BSA reducedthe adhesion of [¹¹¹ In]-labelled platelets to the injured vessels overfour-fold compared to control (71.3 +/- 40.4×10³ cpm/gm, n=6, vs. 16.3+/- 6.2×10³ cpm/gm, n=6, P=0.031).

S-NO-BSA and pS-NO-BSA effects on neointimal proliferation: Neointimalproliferation after local delivery of S-nitrosylated proteins andappropriate controls were evaluated by comparing absolute neointimalarea and neointima/media ratios, and are shown in FIGS. 5A and 5B,respectively The administration of S-NO-BSA (containing 0.3±0.1 molesdisplaceable NO per mole albumin) did not significantly reduceneointimal area (2.54 +/- 0.33×10⁵ μm² vs. 1.83 +/- 0.18×10⁵ μm², n=15)or neointima/media ratio (1.07 +/- 0.167 vs. 0.72 +/- 0.084, n=15) 14days after balloon injury, although a trend was noted. By contrast, theadministration of pS-NO-BSA (containing 3.2±1.3 moles displaceable NOper mole albumin) with a greater displaceable NO content did reduceneointimal area and neointima/media ratio by 81% (2.24 +/- 0.328×10⁵ μm²vs. 0.41 +/- 0.11×10⁵ μm², n=7, P=0.022) and 77% (0.85 +/- 0.122 vs.0.196 +/- 0.66, n=7, P=0.025), respectively. The neointimal area (0.23+/- 0.07×10⁵ μm²) and neointima/media ratio (0.116 +/- 0.041, n=7) inthe sham operated animals were comparable to those of the vesselstreated with pS-NO-BSA. Using relatively high concentrations of aconventional NO donor, SNP (2300 μM), we noted a trend towardsinhibition of neointimal proliferation in both neointimal area (1.47 +/-4.15×10⁵ μM², P=0.056) and neointima/media ratio (0.603 +/- 0.19, n=5,P=0.11) compared to control.

pS-NO-BSA effects on cellular proliferation: Mouse monoclonal antibodystaining against PCNA was used to assay the degree of Sl-phase activityat 7 days after injury. At this time, no difference in the percent ofproliferating cells was noted between vessels treated with pSBSA (30.1%+/- 5.9, n=5) and vessels treated with pS-NO-BSA (37.8% +/- 5.9, n=6).Similarly, no significant difference was noted in the neointimalproliferation of the pS-NO-BSA-treated vessels compared to thepS-BSA-treated controls (neointimal area: 0.124±0.06×10⁵ μM² vs. 0.258+/- 0.19×10⁵ μm², n=5, P=0.54, and neointima/media ratio: 0.032±0.005vs. 0.068±0.027, n=5, Pn=0.15).

Displaceable NO effect on neointimal proliferation: Since S-NO-BSAexhibited a trend toward inhibition and pS-NO-BSA reduced neointimalproliferation, we examined the relationship between the amount ofdisplaceable NO and the extent of neointimal response following vascularinjury, and the results are presented in FIGS. 6A and 6B. There was asignificant inverse relationship between displaceable NO and neointimalproliferation as quantified by absolute neointimal area (P<0.001) (FIG.6A) and the neointima/media area ratio (P<0.001) (FIG. 6B).

pS-NO-BSA treated vessel effect on platelet cGMP and vessel cGMP: NOinhibits platelets and relaxes smooth muscle cells through acGMP-mediated mechanism. We tested the ability of pS-NO-BSA-treatedvessels to deliver NO to platelets, and these results are shown in FIG.7. Platelet cGMP was significantly increased after a one-minute exposureto pS-NO-BSA-treated vessels compared to PS-BSA controls (19.9 +/- 3.3vs 4.11 +/- 0.9 pmol 10⁸ platelets, n=14, P<0.001). In addition, vesselcGMP levels were also elevated after treatment with pS-NO-BSA comparedto PS-BSA control (0.48 +/- 0.46 vs 0.283 +/- 0.23 pmol/mg protein, n=3)suggesting a direct effect on vascular smooth muscle cells, as well.

Discussion

We have previously demonstrated that NO combines with protein sulfhydrylgroups to form stable, biologically active molecules with cGMP-dependentvasodilatory and antiplatelet properties, both in vitro and in vivo(Stamler et al., Proc. Natl. Acad. Sci. U S A., 89:444-448, 1992);(Weldinger et al., Circulation, 81:1667-1679, 1990). The data presentedhere demonstrate that serum albumin, after S-nitrosylation, can bindavidly to balloon-injured femoral arteries and inhibit neointimalproliferation. This phenomenon is associated with diminished plateletdeposition at the site of injury through a cGMP-dependent mechanism.Moreover, the extent of inhibition of neointima formation is directlyrelated to the quantity of displaceable NO carried by albumin.

The endothelium is essential for vascular integrity, control ofthrombosis, (Clowes et al., Lab. Invest. 49:327-333, 1983); (Rees etal., Proc. Natl. Acad. Sci. USA. 86:3375-3378, 1989) and the regulationof intimal growth (Kubes et al., Proc. Natl. Acad. Sci. USA,88:4651-4655, 1991). The endothelium serves these functions by theproduction of locally active effector molecules including EDRF, acompound that has been identified as NO or a closely related molecule.EDRF is responsible, in part, for many biologic actions via theactivation of guanylyl cyclase, including relaxation of vascular smoothmuscle, (Myers et al., Nature (Lond.), 345:161-163, 1990); (Kubes andGranger, Am. J. Physiol. 262:H611-H615, 1993) inhibition of platelets,(Radomski et al., Br. J Pharmacol, 92:181-187, 1987) control ofleukocyte adhesion to the subendothelium, (Reidy, Lab. Invest.,5:513-520, 1985) modulation of vascular permeability, (Groves et al.,Circulation, 87:590-597, 1993) and, perhaps, local control of vascularsmooth muscle growth. Since balloon angioplasty removes the endotheliumfrom arterial smooth muscle, these endothelial functions are lost duringthe procedure. In particular, removal of the endothelium and damage tothe smooth muscle cells are associated with intimal proliferation(McNamara et al., Biochem. Biophys. Res. Commun., 193:291-296, 1993).The mechanism for this response is complex and involves plateletdeposition and activation, cytokine elaboration, smooth muscle cellmigration and proliferation, and extra-cellular matrix production. Afterballoon injury, the endothelium regenerates rapidly but is oftendysfunctional, and presumably unable to maintain an adequateantithrombotic, vasodilating, and antiproliferative phenotype (Saville,Analyst 83:670-672, 1958).

No donors have been used with some success in the setting of ballooninjury to produce decreases in intimal proliferation and in plateletdeposition. In the porcine carotid model, Groves and colleagues (Kubesand Granger, Am. J. Physiol. 262:H611-H615, 1993) demonstrated reducedplatelet adhesion and thrombus formation locally after systemicadministration of SIN-1, a spontaneous NO donor and metabolite ofmolsidomine. These authors showed a 2.3-fold reduction in plateletdeposition without any significant hemodynamic changes. Becauseadministration of this agent was associated with an increase in templatebleeding time and in platelet cGMP, it is possible that SIN-1 exertedits effects through systemic platelet inhibition. A preliminary reportfrom the ACCORD trial also suggests that NO donors might be effectiveadjuncts for balloon angioplasty in humans (The ACCORD StudyInvestigators, J. Am. Coll. Cardiol. 23:59A. (Abstr.), 1994). Thismulticenter study evaluated SIN-1 acutely and molsidomine chronicallyover six months with diltiazem treatment as a control arm in patientsundergoing balloon angioplasty. The loss index and binary restenosisrate were significantly improved in the NO treatment group, although therate loss was not significantly different between groups. Chronicsupplementation with L-arginine, a precursor of endothelium-derivednitric oxide, has been shown to reduce intimal hyperplasia in rabbitthoracic aorta (Cayatte et al., Arterioscler. Thromb., 14:753-9, 1994)and the rat carotid artery (von der Leven et al., Clin. Res., 42:180A.(Abstr.), 1994). By contrast, administration of an inhibitor of NOsynthase, N^(G) -nitro-L-arginine methyl ester, accelerated neointimalformation in the setting of balloon injury (Taubman, wall injury.,Thromb. Haemost., 70:180-183, 1993).

von der Leven and Dzau recently reported (Zeiher et al., Circulation,88:1-367. (Abstr.), 1993) successful transfection of the constitutiveendothelial-type nitric oxide synthase (eNOS) gene in a rat carotidinjury model. In that preliminary study, eNOS incorporation and NOproduction were demonstrable four days after transfection, andneointimal proliferation was partially inhibited two weeks after injuryand transfection. In our study, S-nitrosylated albumin was administeredacutely and, given its half-life of 12 hours, (Benesch and Benesch,Proc. Natl. Acad. Sci. USA, 44:848-853, 1958 it is unlikely thatsignificant amounts of displaceable NO were still present four daysafter injury. The effectiveness of both early and late administration ofNO suggests that NO may influence the complex response to injury bymultiple mechanisms. In addition to modifying the development ofplatelet thrombus and the release of growth factors from platelets,local delivery of S-nitrosothiols could modulate gene transcription invascular smooth muscle cells (Lefer et al., Circulation, 88:1-565.(Abstr.), 1993) as well as smooth muscle metabolism following injury.

Our data demonstrate a profound limitation of neointimal proliferationafter a single, local administration of a durable, potentS-nitrosothiol. Antiplatelet activity may explain these findings, inpart, since we observed a four-fold reduction in platelet deposition toinjured arterial segments after treatment with pS-NO-BSA. Similarly, wealso demonstrated direct platelet inhibition by the pS-NO-BSA-treatedvessel rings. Inhibition of platelet binding would result in manyeffects that are likely to reduce the proliferative response afterinjury. For example, platelet adhesion and aggregation is associatedwith the release of PDGF, basic fibroblast growth factor, epidermalgrowth factor, and transforming growth factor-β, potent stimuli forsmooth muscle cell proliferation and matrix production. pS-NO-BSA couldalso exert its effect by modulating leukocytes though downregulatedexpression of either monocyte chemoattractant protein-i (Hanke et al.,Circ. Res., 67:651-659, 1990) or adhesion molecules (Lefer et al.,Circulation, 88:1-565. (Abstr.), 1993). We cannot exclude a directinhibitory effect of NO on vascular smooth muscle gene expression,migration, proliferation or synthesis of extracellular matrix.

The demonstration of unaltered PCNA-positive cells in vessels treatedwith pS-NO-BSA compared to control vessels is intriguing. Hankedemonstrated significant DNA synthesis in the neointima and media of arabbit carotid model using electrical stimulation. Maximal DNA synthesisoccurred at approximately seven days (Hanke et al., Circ. Res.,67:651-659, 1990) and lasted until at least fourteen days. Ourobservations suggest a mechanism other than the inhibition of local cellreplication by which to explain the inhibition of neointimalproliferation in the rabbit injury model. Such mechanisms could includean early effect on vascular smooth muscle cell migration, transientinhibition of DNA synthesis which is not evident on day seven afterinjury, inhibition of extracellular matrix production, or inhibition ofanother factor(s) required for neointima formation.

These findings have several implications for the treatment of humandisease. Mechanical removal of the endothelium abolishes the vasodilatorresponses to endothelium-dependent vasoactive stimuli, while leaving thevasoconstrictor effects of agonists to smooth muscle unopposed(Furchgott Zawadzki, Nature (Lond.). 288-373-376, 1980). This processoccurs with balloon angioplasty especially at sites where plateletthrombus is noted (Uchida et al., Am. Heart. J., 117:769-776, 1989);(Steele et al., Circ. Res., 57:105-112, 1985). The strategy of localreplacement of an important endothelial product as therapy for acutethrombotic phenomena and restenosis following angioplasty is, thus,suggested by our study.

In summary, our results demonstrate that a stable NO adduct of serumalbumin binds avidly to balloon-injured subendothelium when deliveredlocally. When modified to carry multiple NO groups, pS-NO-BSA markedlydecreases neointimal proliferation after balloon injury. Local deliveryof this molecule decreases platelet adhesion to the injuredsubendothelium and directly inhibits the platelet, interrupting a commonpathway through which growth responses are initiated. These resultssupport the hypothesis that local supplementation of a long-acting NOdonor can favorably modulate vascular injury. The implications of thesefindings suggest that local delivery of S-nitrosothiols may be aneffective treatment for disease states marked by abnormal or absentendothelium, including restenosis after angioplasty.

EXAMPLE 4 Porcine Angiographic Stenosis Model

Pigs were subjected to coronary balloon-injury using standard methods,in accordance with the protocol illustrated in FIG. 8. A perforated drugdelivery balloon catheter was used at the time of balloon injury forinfusion of polythiolated, polynitrosated albumin and with albumincontrol, each of which were infused at a concentration of 1.5 μM for aperiod of 15 minutes. The balloon of the catheter was then deflated andthe catheter was removed. Thereafter, another angiogram was performed todetermine, at 30 minutes after injury, the degree of spasm. Then allcatheters were removed and the incision sites were repaired. The animalswere awakened and maintained with normal chow diets over the next fourweeks. At the end of that period of time, the animals were againsedated, underwent coronary angiography to determine coronary stenosesat the site of angioplasty, after which they were euthanized by anoverdose of pentobarbital. Their coronary arteries perfusion fixed with100 mm Hg of perfusion pressure. They were fixed with formalin,harvested and sectioned for quantitative morphometric assessment of thelumen diameter, the neointimal dimension and cross-section, as well asthe neointimal area. The arteries were stained with hematoxylin andeosin. The neointima to lumen diameter ratio was determined and isillustrated by comparison in FIG. 9.

In this study, a number of normal cholesterolomic pigs were subjected toangioplasty and the effect on their coronary artery was evaluated ingroups which received pS-NO-BSA and which received pS-BSA as a controlor placebo. Four weeks after angioplasty, the animals were sacrificed,their coronary arteries were recovered with perfusion fixation of theartery at autopsy.

FIG. 10 is a histogram which illustrates the diameter (mm) of theneointimal lumen of 14 normocholesterolemic pigs which were subjected toa balloon angioplasty which induced injury of the right coronary artery.Thereafter, they received 1.5 μM pS-NO-BSA or pS-BSA as a control.

This measurement was made at four weeks into the protocol by coronaryangiography. The animals were sedated, catheters were placed in thecoronary ostea and radiocontrast fluid was infused. The angiograms wererecorded and subsequently processed by a computer-driven quantitativecoronary angiography algorithm to determine precisely the lumendiameter. The degree of stenosis represents the percentage reduction inthe lumen diameter compared with a reference segment proximal to thearea of stenosis using standard methods. The results of this areillustrated in FIG. 10.

FIG. 10 is a histogram which illustrates a degree of coronary stenosisobserved at four weeks after angioplasty in pigs which received 1.5 μMpS-NO-BSA or pS-BSA as a control.

This measurement was made during the initial balloon injury procedure.Within 30 minutes following the procedure, the animals underwentcoronary angiography, coronary catheters were placed in the coronaryostea, radiocontrast was infused into the coronary arteries andmeasurements were made of the degree of so-called "recoil spasm" thatexisted at the point of angioplasty. The degree of spasm or recoil wasdefined quantitatively, again using the computer-driven quantitativecoronary angiography algorithm that compared the segment at the site ofballoon injury with a proximal segment that was uninjured as a referencestandard. The results are illustrated graphically in FIG. 11.

FIG. 11 is a histogram which illustrates the extent of coronary spasminduced distal the site of injury as compared to the pre-existing baseline in pigs which received 1.5 μM pS-NO-BSA or pS-BSA as a control.

Next, a quantitative measurement was made by morphometric assessmentfollowing autopsy and after perfusion fixation of the vessel todetermine lumen diameter at four weeks. The results are illustrated inFIG. 12.

FIG. 12 is a histogram which illustrates the inner-diameter of the lumenof the right coronary artery of pigs four weeks after they received 1.5μM pS-NO-BSA or pS-BSA as a control.

EXAMPLE 5 Coating Palmaz-Schatz Stents With pS NO-BSA

The experiments recorded here were performed in order to determine:whether this pS NO-BSA would adhere to the metallic surface of aPalmaz-Schatz stent; whether there would be enough nitric oxideavailable to inhibit platelet adhesion and aggregation near the metallicsurface; whether coating of a Palmaz-Schatz stent with pS NO-BSA wouldsignificantly reduce the deposition of Indium¹¹¹ labeled platelets whenplaced in the carotid arteries of pigs; whether coating a Palmaz-Schatzstent would decrease the degree of anticoagulation needed to maintainpatency; and whether the coating would reduce the degree and severity ofneointimal hyperplasia leading to restenosis.

Palmaz-Schatz stents were dip-coated in 800-1000 μM SNO-BSA three timesfor 10 minutes followed by 10 minutes of air drying time. Then, one weeklater, three coated stents were immersed in platelet rich plasma (PRP)for 2 minutes. A control uncoated stent was also immersed in anotheraliquot of the same PRP.

The increase in platelet cyclic GMP levels was determined and is shownin Table 1.

                  TABLE 1                                                         ______________________________________                                        Cyclic GMP Levels in PRP Exposed for 2 Minutes                                to pS NO-BSA Coated and Uncoated Palmaz-Schatz Stents                                        (P moles CGMP/10.sup.8 platelets)                              Conc of NO (Saville R.sub.x)                                                                   A       B      C     D                                       ______________________________________                                        500 μM        7.2     6.8    6.9   6.3                                                      7.0     6.0    6.8   4.6                                                      2.8     2.8    2.4   1.7                                     800 μm        5.8     5.6    5.9   3.9                                                      10.4    11.0   10.8  8.5                                     1000 μm       4.6     4.9    5.7   1.9                                                      6.6     5.9    6.3   3.0                                                      5.9     5.1    5.7   2.9                                     ______________________________________                                    

The three columns on the left (columns A through C) show the levels ofcGMP in the platelets which were exposed to a coated stent and thecolumn on the right (Column D) shows the level of cyclic GMP in the samePRP which was exposed to the uncoated stent.

A coated and an uncoated stent were placed in the carotid arteries ofpigs, one in each carotid artery. Then Indium¹¹¹ labeled platelets werecirculated for four hours. At the end of the four hours, the arteriescontaining the stents were removed and placed in a Gamma counter well.The counts on stents indicate the degree of platelet deposition on eachstent. The data is shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Indium.sup.III Labeled Platelet Counts                                        on S-No-BSA Coated Versus Uncoated                                            Palmaz-Schatz Stents                                                          P S NO-BSA Coated  Uncoated Ratio                                             ______________________________________                                        59,760             1,076,300                                                                              18                                                94,000             246,000  2.6                                               126,400            868,600  6.0                                               61,500             347,400  5.7                                               120,000            684,000  5.6                                               88,600             264,462  3.0                                               135,000            590,000  4.1                                               14,160              43,900  3.2                                               ______________________________________                                    

One coated and one uncoated stent was placed in each of the two carotidarteries under sterile conditions in 10 pigs. They will be followed for28 days and then the stented carotid arteries will be removed. They willbe examined histologically for the degree of neointimal hyperplasia.

What is claimed is:
 1. A method for preventing adverse effectsassociated with the use of a medical device in a patient comprisingintroducing into said patient a medical device of which at least aportion includes a nitric oxide adduct, wherein said nitric oxide adductcomprises a sodium nitroprusside, a nitrosothiol, a nitrate, a nitriteor a nitrosated amino acid.
 2. The method of claim 1 wherein the nitricoxide adduct is present in a matrix coating on a surface of the medicaldevice which contacts a tissue within the patient, wherein the matrixcoating is nylon or plastic.
 3. The method of claim 2 wherein the matrixcoating provides for release of the intact nitric oxide adduct.
 4. Themethod of claim 3 wherein the matrix coating provides for the sustainedrelease of the nitric oxide adduct.
 5. The method of claim 1 wherein thenitric oxide adduct is coated per se on a surface of the medical devicewhich contacts the vessel at the site of injury.
 6. The method of claim1 wherein the nitric oxide adduct is directly or indirectly bound toreactive sites on a surface of the medical device which contacts thevessel at the site of injury.
 7. The method of claim 1 wherein at leasta portion of the medical device is formed of a polymer which includesthe nitric oxide adduct, wherein the polymer is nylon, polyethyleneperthalate or polytetrafluoroethylene.
 8. The method of a claim 7wherein the material is a polymer.
 9. The method of claim 8 wherein thepolymer is selected from the group consisting of nylon, polyethyleneperthalate and polytetrafluoroethylene.
 10. The method of claim 7wherein the material provides for release of the intact nitric oxideadduct.
 11. The method of claim 7 wherein the material provides for thesustained release of the nitric oxide adduct.
 12. The method of claim 7wherein the material is biodegradable.
 13. The method of claim 12wherein the material is bioresorbable.
 14. The method of claim 1 whereinsaid medical device comprises a catheter, prosthetic heart valve,synthetic vessel graft, stent, arteriovenous shunt or artificial heart.15. The method of claim 1 which comprises administering the nitric oxideadduct in combination with an anti-thrombogenic agent.
 16. The method ofclaim 15 wherein said anti-thrombogenic compound comprises heparin,hirudin, analogs of hirudin warfarin, aspirin, indomethacin,dipyridamole, prostacyclin, prostaglandin-E, sulfinpyrazone,phenothiazines, RGD peptide, RGD peptide mimetics, agents that blockplatelet glycoprotein IIb-IIIa receptors, ticlopidine or clopidogrel.17. A method for preventing adverse effects associated with the use of amedical device in a patient comprising introducing into said patient amedical device during a medical procedure and before or during saidprocedure locally administering a nitric oxide adduct to the site ofcontact of said device with any internal tissue, wherein said nitricoxide adduct comprises a sodium nitroprusside, a nitrosothiol, a nitriteor a nitrosylated amino acid.
 18. The method of claim 17 wherein saidnitric oxide adduct is administered through the lumen of anintraarterial or intravenous catheter.
 19. A medical device comprisingan instrument suitable for introduction into a patient of which at leasta portion comprises a nitric oxide adduct, wherein said nitric oxideadduct comprises a sodium nitroprusside, a nitrosothiol, a nitrate, anitrite or a nitrosated amino acid.
 20. The medical device of claim 19wherein the nitric oxide adduct is present in a matrix coating on asurface of the instrument which contacts a tissue within the patient,wherein the matrix coating is nylon or plastic.
 21. The device of claim20 wherein the matrix coating provides for release of the intact nitricoxide adduct.
 22. The device of claim 21 wherein the matrix coatingprovides for the sustained release of the nitric oxide adduct.
 23. Thedevice of claim 19 wherein the nitric oxide adduct is coated per se on asurface of the medical device which contacts the vessel at the site ofinjury.
 24. The device of claim 19 wherein the nitric oxide adduct isdirectly or indirectly bound to reactive sites on a surface of theinstrument which contacts the vessel at site of injury.
 25. The deviceof claim 19 wherein at least a portion of the instrument is formed of apolymer which includes the nitric oxide adduct, wherein the polymer isnylon, polyethylene perthalate or polytetrafluroethylene.
 26. The deviceof claim 25 wherein the material is a polymer.
 27. The device of claim26 wherein the polymer is selected from the group consisting of nylon,polyethylene perthalate and polytetrafluoruethylene.
 28. The device ofclaim 25 wherein the material provides for release of the intact nitricoxide adduct.
 29. The device of claim 25 wherein the material providesfor the sustained release of the nitric oxide adduct.
 30. The device ofclaim 25 wherein the material is biodegradable.
 31. The device of claim30 wherein the material is bioresorbable.
 32. The device of claim 19wherein said medical device comprises a catheter, prosthetic heartvalve, synthetic vessel graft, stent, arteriovenous shunt or artificialheart.
 33. A method for treating a damaged vessel in a patient in needthereof which comprises introducing into said vessel at the site ofdamage the medical device of claim
 19. 34. A method for treating adamaged vessel in a patient in need thereof which comprises introducinginto said vessel at the site of damage the medical device of claim 20.35. A method for treating a damaged vessel in a patient in need thereofwhich comprises introducing into said vessel at the site of damage themedical device of claim
 23. 36. A method for treating a damaged vesselin a patient in need thereof which comprises introducing into saidvessel at the site of damage the medical device of claim
 24. 37. Amethod for treating a damaged vessel in a patient in need thereof whichcomprises introducing into said vessel at the site of damage the medicaldevice of claim
 25. 38. A method for treating a damaged vessel in apatient in need thereof which comprises introducing into said vessel atthe site of damage the medical device of claim
 32. 39. The method ofclaim 1, wherein said nitrosothiol is a long carbon chain lipophilicnitrosothiol.
 40. The method of claim 1, wherein said nitrosothiol is anitrosodithiol.
 41. The method of claim 17, wherein said nitrosothiol isa long carbon chain lipophilic nitrosothiol.
 42. The method of claim 17,wherein said nitrosothiol is a nitrosodithiol.
 43. The device of claim19, wherein said nitrosothiol is a long carbon chain lipophilicnitrosothiol.
 44. The device of claim 19, wherein said nitrosothiol is anitrosodithiol.
 45. The method of claim 1, wherein said nitric oxideadduct delivers a nitrosonium ion or a nitroxyl ion under physiologicalconditions.
 46. The method of claim 17, wherein said nitric oxide adductdelivers a nitrosonium ion or a nitroxyl ion under physiologicalconditions.
 47. The device of claim 19, wherein said nitric oxide adductdelivers a nitrosonium ion or a nitroxyl ion under physiologicalconditions.
 48. The method of claim 17, further comprising administeringsaid nitric oxide adduct in combination with an anti-thrombogeniccompound.
 49. The method of claim 48, wherein said anti-thrombogeniccompound comprises heparin, hirudin, an analog of hirudin, warfarin,aspirin, indomethacin, dipyridamole, prostacyclin, prostaglandin-E,sulfinpyrazone, phenothiazine, RGD peptide, RGD peptide mimetic, agentsthat block platelet glycoprotein IIb-IIIa receptors, ticlopidine orclopidogrel.
 50. The device of claim 19, wherein said device furthercomprises an anti-thrombogenic compound.
 51. The device of claim 50,wherein said anti-thrombogenic compound comprises heparin, hirudin, ananalog of hirudin, warfarin, aspirin, indomethacin, dipyridamole,prostacyclin, prostaglandin-E, sulfinpyrazone, phenothiazine, RGDpeptide, RGD peptide mimetic, agents that block platelet glycoproteinIIb-IIIa receptors, ticlopidine or clopidogrel.
 52. The method of claim1, wherein said nitrate is a thionitrate.
 53. The method of claim 1,wherein said nitrite is a thionitrite.
 54. The method of claim 17,wherein said nitrate is a thionitrate.
 55. The method of claim 17,wherein said nitrite is a thionitrite.
 56. The device of claim 19,wherein said nitrate is a thionitrate.
 57. The device of claim 19,wherein said nitrite is a thionitrite.